{"gene":"PRSS54","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2022,"finding":"PRSS54 localizes to the acrosomal granule during early acrosome biogenesis, then extends along the inner acrosomal membrane, and is present in the acrosome region of mature sperm; loss of PRSS54 causes acrosome deformation, unfused vacuoles, detachment and eccentric localization of acrosomal granules, asymmetrical nuclear elongation, defective sperm-zona penetration, and male subfertility—all rescued by Prss54 transgene.","method":"CRISPR/Cas9 knockout, transgenic rescue, transmission electron microscopy, subcellular localization imaging, fertility assays","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotype, transgenic rescue, and TEM ultrastructure; single lab but multiple orthogonal methods","pmids":["35863763"],"is_preprint":false},{"year":2022,"finding":"PRSS54 physically interacts with acrosomal proteins ZPBP1, ZPBP2, ACRBP, and ZP3R, and loss of PRSS54 disrupts the distribution (but not protein levels) of these interactors in testis and sperm.","method":"Co-immunoprecipitation (interaction), immunofluorescence (distribution analysis in Prss54-/- vs. control)","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP/pulldown with distribution phenotype, single lab","pmids":["35863763"],"is_preprint":false},{"year":2022,"finding":"PRSS54 protein undergoes processing from ~50 kDa in testis to ~42 kDa in mature sperm, indicating proteolytic maturation during spermatogenesis.","method":"Western blot of testis vs. sperm protein extracts","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 3 — single biochemical observation, single lab","pmids":["35863763"],"is_preprint":false},{"year":2022,"finding":"Prss54-/- sperm show hypersensitivity to acrosome reaction inducers, indicating PRSS54 regulates acrosomal stability.","method":"Acrosome reaction induction assay in Prss54-/- vs. control sperm","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined functional readout, single lab","pmids":["35863763"],"is_preprint":false},{"year":2020,"finding":"Individual ablation of Prss54 by CRISPR/Cas9 in mice yields homozygous knockout males with no significant differences in testis weight, testis/epididymis histology, sperm morphology, or fertility compared to controls, indicating PRSS54 is not individually required for male fertility (or functions redundantly).","method":"CRISPR/Cas9 knockout, histology, sperm morphology analysis, fertility assay","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with multiple phenotypic readouts; discrepancy with later study may reflect strain or experimental differences","pmids":["31403672"],"is_preprint":false},{"year":2024,"finding":"PRSS54 is among the proteins that interact with SPEM2, and SPEM2 is required for proper processing and maturation of PRSS54 in epididymal sperm.","method":"Co-immunoprecipitation (SPEM2 interactome), Western blot of Spem2-/- epididymal sperm","journal":"Cellular and molecular life sciences : CMLS","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP from another gene's study; PRSS54 is a secondary finding","pmids":["38421455"],"is_preprint":false}],"current_model":"PRSS54 is a testis-specific serine protease that localizes sequentially to the acrosomal granule and inner acrosomal membrane during spermiogenesis; it undergoes proteolytic maturation (50→42 kDa) and interacts with acrosomal proteins ZPBP1, ZPBP2, ACRBP, and ZP3R to regulate acrosomal granule positioning, sperm head morphogenesis, and zona pellucida penetration, while also being subject to SPEM2-dependent processing in the epididymis."},"narrative":{"teleology":[{"year":2020,"claim":"An initial CRISPR knockout asked whether PRSS54 is individually required for male fertility: single-gene ablation revealed no detectable fertility or morphological defect, suggesting functional redundancy among testicular serine proteases.","evidence":"CRISPR/Cas9 knockout mice with histology, sperm morphology, and fertility assays","pmids":["31403672"],"confidence":"Medium","gaps":["Discrepancy with later knockout findings unresolved — may reflect strain background or assay sensitivity differences","No ultrastructural analysis was performed","Redundancy partners not identified"]},{"year":2022,"claim":"A comprehensive knockout study resolved PRSS54's cellular function, showing it is required for acrosomal granule positioning, membrane fusion during acrosome biogenesis, nuclear shaping, and sperm-zona penetration — establishing PRSS54 as a key acrosomal organizer rather than a redundant protease.","evidence":"CRISPR/Cas9 knockout with transgenic rescue, TEM ultrastructure, immunofluorescence localization, fertility and acrosome reaction assays in mice","pmids":["35863763"],"confidence":"High","gaps":["Catalytic activity of PRSS54 not demonstrated — whether protease activity or scaffolding function underlies the phenotype is unknown","The basis for discrepancy with the 2020 no-phenotype knockout remains unexplained","Direct substrates of PRSS54 protease activity not identified"]},{"year":2022,"claim":"Identification of ZPBP1, ZPBP2, ACRBP, and ZP3R as physical interactors whose acrosomal distribution depends on PRSS54 established the molecular network through which PRSS54 organizes the acrosome.","evidence":"Co-immunoprecipitation and immunofluorescence redistribution analysis in Prss54−/− versus control testes and sperm","pmids":["35863763"],"confidence":"Medium","gaps":["Interactions demonstrated by single Co-IP without reciprocal pulldown validation","Whether interactions are direct or bridged by other acrosomal components is unknown","Functional consequence of each individual interaction not dissected"]},{"year":2024,"claim":"PRSS54 was identified as a SPEM2-interacting protein whose epididymal processing depends on SPEM2, linking PRSS54 maturation to an epididymal quality-control pathway.","evidence":"Co-immunoprecipitation of SPEM2 interactome and Western blot of Spem2−/− epididymal sperm","pmids":["38421455"],"confidence":"Low","gaps":["PRSS54 was a secondary finding in a SPEM2-focused study; awaits dedicated validation","Whether impaired PRSS54 processing in Spem2−/− sperm contributes to fertility defects is untested","Nature of SPEM2-dependent cleavage event not characterized"]},{"year":null,"claim":"It remains unknown whether PRSS54's biological role depends on its serine protease catalytic activity or on a non-enzymatic scaffolding function, and no direct proteolytic substrates have been identified.","evidence":"","pmids":[],"confidence":"High","gaps":["No catalytic-dead mutant rescue experiment performed","No in vitro protease activity assay reported","Human relevance for male infertility not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0,3]}],"complexes":[],"partners":["ZPBP1","ZPBP2","ACRBP","ZP3R","SPEM2"],"other_free_text":[]},"mechanistic_narrative":"PRSS54 is a testis-specific serine protease essential for acrosome biogenesis, sperm head morphogenesis, and zona pellucida penetration during mammalian spermatogenesis. During spermiogenesis, PRSS54 localizes first to the acrosomal granule and subsequently extends along the inner acrosomal membrane; its loss causes acrosomal granule detachment, unfused vacuoles, asymmetrical nuclear elongation, acrosomal instability, and male subfertility, all of which are rescued by transgenic re-expression [PMID:35863763]. PRSS54 physically interacts with acrosomal proteins ZPBP1, ZPBP2, ACRBP, and ZP3R, and its absence disrupts their spatial distribution without altering their protein levels, indicating a scaffolding or organizational role within the acrosome [PMID:35863763]. PRSS54 undergoes proteolytic maturation from ~50 kDa in testis to ~42 kDa in mature sperm, a process that depends in part on the epididymal protein SPEM2 [PMID:35863763, PMID:38421455]."},"prefetch_data":{"uniprot":{"accession":"Q6PEW0","full_name":"Inactive serine protease 54","aliases":["Cancer/testis antigen 67","CT67","Plasma kallikrein-like protein 4"],"length_aa":395,"mass_kda":43.8,"function":"","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q6PEW0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PRSS54","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/PRSS54","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in single","driving_tissues":[{"tissue":"testis","ntpm":38.7}],"url":"https://www.proteinatlas.org/search/PRSS54"},"hgnc":{"alias_symbol":["FLJ25339","KLKBL4","CT67"],"prev_symbol":[]},"alphafold":{"accession":"Q6PEW0","domains":[{"cath_id":"2.40.10.10","chopping":"55-272","consensus_level":"medium","plddt":84.6191,"start":55,"end":272}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6PEW0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6PEW0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6PEW0-F1-predicted_aligned_error_v6.png","plddt_mean":67.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PRSS54","jax_strain_url":"https://www.jax.org/strain/search?query=PRSS54"},"sequence":{"accession":"Q6PEW0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6PEW0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6PEW0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6PEW0"}},"corpus_meta":[{"pmid":"31403672","id":"PMC_31403672","title":"The testis-specific serine proteases PRSS44, PRSS46, and PRSS54 are dispensable for male mouse fertility†.","date":"2020","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/31403672","citation_count":42,"is_preprint":false},{"pmid":"15915581","id":"PMC_15915581","title":"Stepwise hydration of ionized aromatics. Energies, structures of the hydrated benzene cation, and the mechanism of deprotonation reactions.","date":"2005","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/15915581","citation_count":38,"is_preprint":false},{"pmid":"35863763","id":"PMC_35863763","title":"Testis-specific serine protease PRSS54 regulates acrosomal granule localization and sperm head morphogenesis in mice†.","date":"2022","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/35863763","citation_count":13,"is_preprint":false},{"pmid":"38421455","id":"PMC_38421455","title":"Spem2, a novel testis-enriched gene, is required for spermiogenesis and fertilization in mice.","date":"2024","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/38421455","citation_count":12,"is_preprint":false},{"pmid":"39208330","id":"PMC_39208330","title":"Differential expression and regulation of ADAD1, DMRTC2, PRSS54, SYCE1, SYCP1, TEX101, TEX48, and TMPRSS12 gene profiles in colon cancer tissues and their in vitro response to epigenetic drugs.","date":"2024","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/39208330","citation_count":5,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4280,"output_tokens":1303,"usd":0.016192},"stage2":{"model":"claude-opus-4-6","input_tokens":4543,"output_tokens":1460,"usd":0.088822},"total_usd":0.105014,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2022,\n      \"finding\": \"PRSS54 localizes to the acrosomal granule during early acrosome biogenesis, then extends along the inner acrosomal membrane, and is present in the acrosome region of mature sperm; loss of PRSS54 causes acrosome deformation, unfused vacuoles, detachment and eccentric localization of acrosomal granules, asymmetrical nuclear elongation, defective sperm-zona penetration, and male subfertility—all rescued by Prss54 transgene.\",\n      \"method\": \"CRISPR/Cas9 knockout, transgenic rescue, transmission electron microscopy, subcellular localization imaging, fertility assays\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype, transgenic rescue, and TEM ultrastructure; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"35863763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PRSS54 physically interacts with acrosomal proteins ZPBP1, ZPBP2, ACRBP, and ZP3R, and loss of PRSS54 disrupts the distribution (but not protein levels) of these interactors in testis and sperm.\",\n      \"method\": \"Co-immunoprecipitation (interaction), immunofluorescence (distribution analysis in Prss54-/- vs. control)\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP/pulldown with distribution phenotype, single lab\",\n      \"pmids\": [\"35863763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PRSS54 protein undergoes processing from ~50 kDa in testis to ~42 kDa in mature sperm, indicating proteolytic maturation during spermatogenesis.\",\n      \"method\": \"Western blot of testis vs. sperm protein extracts\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single biochemical observation, single lab\",\n      \"pmids\": [\"35863763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Prss54-/- sperm show hypersensitivity to acrosome reaction inducers, indicating PRSS54 regulates acrosomal stability.\",\n      \"method\": \"Acrosome reaction induction assay in Prss54-/- vs. control sperm\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined functional readout, single lab\",\n      \"pmids\": [\"35863763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Individual ablation of Prss54 by CRISPR/Cas9 in mice yields homozygous knockout males with no significant differences in testis weight, testis/epididymis histology, sperm morphology, or fertility compared to controls, indicating PRSS54 is not individually required for male fertility (or functions redundantly).\",\n      \"method\": \"CRISPR/Cas9 knockout, histology, sperm morphology analysis, fertility assay\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple phenotypic readouts; discrepancy with later study may reflect strain or experimental differences\",\n      \"pmids\": [\"31403672\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRSS54 is among the proteins that interact with SPEM2, and SPEM2 is required for proper processing and maturation of PRSS54 in epididymal sperm.\",\n      \"method\": \"Co-immunoprecipitation (SPEM2 interactome), Western blot of Spem2-/- epididymal sperm\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP from another gene's study; PRSS54 is a secondary finding\",\n      \"pmids\": [\"38421455\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PRSS54 is a testis-specific serine protease that localizes sequentially to the acrosomal granule and inner acrosomal membrane during spermiogenesis; it undergoes proteolytic maturation (50→42 kDa) and interacts with acrosomal proteins ZPBP1, ZPBP2, ACRBP, and ZP3R to regulate acrosomal granule positioning, sperm head morphogenesis, and zona pellucida penetration, while also being subject to SPEM2-dependent processing in the epididymis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PRSS54 is a testis-specific serine protease essential for acrosome biogenesis, sperm head morphogenesis, and zona pellucida penetration during mammalian spermatogenesis. During spermiogenesis, PRSS54 localizes first to the acrosomal granule and subsequently extends along the inner acrosomal membrane; its loss causes acrosomal granule detachment, unfused vacuoles, asymmetrical nuclear elongation, acrosomal instability, and male subfertility, all of which are rescued by transgenic re-expression [PMID:35863763]. PRSS54 physically interacts with acrosomal proteins ZPBP1, ZPBP2, ACRBP, and ZP3R, and its absence disrupts their spatial distribution without altering their protein levels, indicating a scaffolding or organizational role within the acrosome [PMID:35863763]. PRSS54 undergoes proteolytic maturation from ~50 kDa in testis to ~42 kDa in mature sperm, a process that depends in part on the epididymal protein SPEM2 [PMID:35863763, PMID:38421455].\",\n  \"teleology\": [\n    {\n      \"year\": 2020,\n      \"claim\": \"An initial CRISPR knockout asked whether PRSS54 is individually required for male fertility: single-gene ablation revealed no detectable fertility or morphological defect, suggesting functional redundancy among testicular serine proteases.\",\n      \"evidence\": \"CRISPR/Cas9 knockout mice with histology, sperm morphology, and fertility assays\",\n      \"pmids\": [\"31403672\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Discrepancy with later knockout findings unresolved — may reflect strain background or assay sensitivity differences\",\n        \"No ultrastructural analysis was performed\",\n        \"Redundancy partners not identified\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A comprehensive knockout study resolved PRSS54's cellular function, showing it is required for acrosomal granule positioning, membrane fusion during acrosome biogenesis, nuclear shaping, and sperm-zona penetration — establishing PRSS54 as a key acrosomal organizer rather than a redundant protease.\",\n      \"evidence\": \"CRISPR/Cas9 knockout with transgenic rescue, TEM ultrastructure, immunofluorescence localization, fertility and acrosome reaction assays in mice\",\n      \"pmids\": [\"35863763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Catalytic activity of PRSS54 not demonstrated — whether protease activity or scaffolding function underlies the phenotype is unknown\",\n        \"The basis for discrepancy with the 2020 no-phenotype knockout remains unexplained\",\n        \"Direct substrates of PRSS54 protease activity not identified\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of ZPBP1, ZPBP2, ACRBP, and ZP3R as physical interactors whose acrosomal distribution depends on PRSS54 established the molecular network through which PRSS54 organizes the acrosome.\",\n      \"evidence\": \"Co-immunoprecipitation and immunofluorescence redistribution analysis in Prss54−/− versus control testes and sperm\",\n      \"pmids\": [\"35863763\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Interactions demonstrated by single Co-IP without reciprocal pulldown validation\",\n        \"Whether interactions are direct or bridged by other acrosomal components is unknown\",\n        \"Functional consequence of each individual interaction not dissected\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"PRSS54 was identified as a SPEM2-interacting protein whose epididymal processing depends on SPEM2, linking PRSS54 maturation to an epididymal quality-control pathway.\",\n      \"evidence\": \"Co-immunoprecipitation of SPEM2 interactome and Western blot of Spem2−/− epididymal sperm\",\n      \"pmids\": [\"38421455\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"PRSS54 was a secondary finding in a SPEM2-focused study; awaits dedicated validation\",\n        \"Whether impaired PRSS54 processing in Spem2−/− sperm contributes to fertility defects is untested\",\n        \"Nature of SPEM2-dependent cleavage event not characterized\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown whether PRSS54's biological role depends on its serine protease catalytic activity or on a non-enzymatic scaffolding function, and no direct proteolytic substrates have been identified.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No catalytic-dead mutant rescue experiment performed\",\n        \"No in vitro protease activity assay reported\",\n        \"Human relevance for male infertility not established\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ZPBP1\", \"ZPBP2\", \"ACRBP\", \"ZP3R\", \"SPEM2\"],\n    \"other_free_text\": []\n  }\n}\n```"}