{"gene":"KPNA5","run_date":"2026-04-28T18:30:27","timeline":{"discoveries":[{"year":2014,"finding":"Crystal structure of the human KPNA5 C-terminus in complex with Ebola virus VP24 (eVP24) revealed that eVP24 recognizes a unique nonclassical NLS binding site on KPNA5 that is necessary for efficient PY-STAT1 nuclear transport. eVP24 binds KPNA5 with very high affinity at this site to competitively inhibit PY-STAT1 nuclear import without affecting classical NLS cargo transport.","method":"X-ray crystallography of KPNA5 C-terminus/eVP24 complex; binding affinity measurements; cell-based nuclear transport assays","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus functional validation with mutagenesis and transport assays in a highly cited study","pmids":["25121748"],"is_preprint":false},{"year":2015,"finding":"Lloviu virus VP24 (LLOV VP24) blocks tyrosine-phosphorylated STAT1 (PY-STAT1) binding to KPNA5, preventing STAT1 nuclear accumulation and IFN-induced gene expression, demonstrating that KPNA5's role in PY-STAT1 nuclear import is targeted by multiple filovirus VP24 proteins.","method":"Co-immunoprecipitation; nuclear accumulation assays; IFN-induced gene expression assays in human and bat cells","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal functional assays in multiple cell types, single lab","pmids":["26255028"],"is_preprint":false},{"year":2015,"finding":"PHB2 interacts with KPNA1, KPNA5, and KPNA6, and these interactions mediate estrogen-dependent translocation of PHB2 into the nuclei of breast cancer cells. BIG3 blocks the KPNA-binding region(s) of PHB2, thereby inhibiting KPNA-mediated PHB2 nuclear translocation.","method":"Co-immunoprecipitation; siRNA knockdown of individual KPNAs; nuclear translocation assays in breast cancer cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus siRNA knockdown with translocation readout, single lab","pmids":["26052702"],"is_preprint":false},{"year":2017,"finding":"VP24 proteins from different Ebolavirus species (EBOV, BDBV, RESTV) all bind KPNA1, KPNA5, and KPNA6, but with differing affinities; BDBV VP24 shows ~10-fold lower KPNA5 binding affinity than EBOV or RESTV VP24. Reduced VP24-KPNA binding affinity decreases IFN inhibition and shortens VP24 half-life, revealing that KPNA interaction stabilizes VP24.","method":"Binding affinity measurements with purified proteins; cell-based VP24 half-life assays; IFN-induced gene expression assays; alanine-scanning mutagenesis","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1-2 — quantitative binding assays with purified proteins plus multiple functional readouts and mutagenesis","pmids":["27974555"],"is_preprint":false},{"year":2017,"finding":"Molecular dynamics simulations of the VP24-KPNA5 complex identified key hydrogen bonds (D480-T138, E483-R137, D205-R396) critical for the interaction interface. VP24 residues R137 and T138, and KPNA5 residues R396, R398, R480, Y477, and F484, were identified as hotspot residues essential for complex stability.","method":"All-atom molecular dynamics simulation; MM-GB/SA energy calculation; computational alanine scanning","journal":"Molecular bioSystems","confidence":"Low","confidence_rationale":"Tier 4 — computational prediction only, no experimental validation","pmids":["28418440"],"is_preprint":false},{"year":2017,"finding":"Macrocyclic peptide inhibitors of the VP24-KPNA5 protein-protein interaction were identified with dissociation constants in the single-digit nanomolar range for eVP24, and these peptides successfully disrupted the eVP24-KPNA interaction, providing the first chemical probes capable of modulating this PPI.","method":"RaPID (Random non-standard Peptides Integrated Discovery) system; binding affinity measurements; PPI disruption assays with purified proteins","journal":"Organic & biomolecular chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro reconstitution of PPI disruption with purified components, single lab","pmids":["28574091"],"is_preprint":false},{"year":2025,"finding":"miR-153 directly targets two binding sites in the 3'UTR of KPNA5 mRNA to suppress KPNA5 expression. KPNA5 facilitates nuclear translocation of NRF2 by binding to three nuclear localization sequences (NLS) of NRF2, and miR-153-mediated suppression of KPNA5 impairs NRF2-mediated antioxidant responses, promoting oxidative stress and cognitive deficits in chronic cerebral hypoperfusion models.","method":"Luciferase reporter assays for miR-153 binding sites; Co-IP for KPNA5-NRF2 interaction; knockdown and overexpression in vivo (rat basal forebrain) and in vitro; nuclear fractionation assays","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus in vivo knockdown with defined cognitive/oxidative phenotype, single lab","pmids":["40816766"],"is_preprint":false},{"year":2025,"finding":"KPNA5 recognizes nuclear localization signals (NLS) in the protein tyrosine phosphatase PTPN4 and mediates its nuclear transport. Nuclear PTPN4 inhibits STAT3 phosphorylation and its downstream signaling pathway. KPNA5 overexpression suppressed ovarian cancer cell proliferation, tumor growth, and invasion in vitro and in vivo.","method":"Co-IP for KPNA5-PTPN4 interaction; Western blot for STAT3 phosphorylation; xenograft tumor model; CCK-8, colony formation, Transwell assays","journal":"Cancer medicine","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional cellular and in vivo assays with defined pathway readout, single lab","pmids":["40145330"],"is_preprint":false},{"year":2025,"finding":"miR-153-3p derived from bone marrow mesenchymal stem cell exosomes targets KPNA5, and this targeting promotes M2 macrophage polarization to facilitate wound healing.","method":"Target prediction and validation; co-culture assays; exosome transfer experiments; macrophage polarization assays","journal":"Applied biochemistry and biotechnology","confidence":"Low","confidence_rationale":"Tier 3 — single lab, limited mechanistic follow-up on KPNA5 specifically","pmids":["40053206"],"is_preprint":false},{"year":2025,"finding":"Nuclear trafficking of eVP24 by KPNA5 requires simultaneous binding of cellular NLS-containing cargo; eVP24 binding to KPNA5 does not impair cargo NLS binding at the major NLS binding site, indicating that eVP24 and classical cargo can simultaneously associate with KPNA5 for co-transport into the nucleus.","method":"Hydrogen-deuterium exchange mass spectrometry (HDX-MS); native MS; biophysical binding assays with purified proteins; nuclear localization assays","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 1-2 — multiple orthogonal biophysical methods with purified proteins, single lab","pmids":["40872766"],"is_preprint":false}],"current_model":"KPNA5 is a nuclear import receptor (NPI-1 subfamily karyopherin alpha) that transports cargo bearing nonclassical NLS sequences—including phosphorylated STAT1 and NRF2—into the nucleus via a unique binding site on its C-terminus; Ebola virus VP24 exploits this site with high affinity to selectively block PY-STAT1 nuclear import while permitting simultaneous binding of classical NLS cargo, and KPNA5 also mediates nuclear import of PTPN4 (suppressing STAT3 signaling) and PHB2 (suppressing ERα signaling), with its activity regulated post-transcriptionally by miR-153 targeting its 3'UTR."},"narrative":{"teleology":[],"mechanism_profile":null,"mechanistic_narrative":"Parse failed — see logs"},"prefetch_data":{"uniprot":{"accession":"O15131","full_name":"Importin subunit alpha-6","aliases":["Karyopherin subunit alpha-5"],"length_aa":539,"mass_kda":60.7,"function":"Functions in nuclear protein import as an adapter protein for nuclear receptor KPNB1. Binds specifically and directly to substrates containing either a simple or bipartite NLS motif. Docking of the importin/substrate complex to the nuclear pore complex (NPC) is mediated by KPNB1 through binding to nucleoporin FxFG repeats and the complex is subsequently translocated through the pore by an energy requiring, Ran-dependent mechanism. At the nucleoplasmic side of the NPC, Ran binds to importin-beta and the three components separate and importin-alpha and -beta are re-exported from the nucleus to the cytoplasm where GTP hydrolysis releases Ran from importin. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus. Mediates nuclear import of STAT1 homodimers and STAT1/STAT2 heterodimers by recognizing non-classical NLSs of STAT1 and STAT2 through ARM repeats 8-9. Recognizes influenza A virus nucleoprotein through ARM repeat 7-9 In vitro, mediates the nuclear import of human cytomegalovirus UL84 by recognizing a non-classical NLS","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O15131/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KPNA5","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":[{"gene":"KPNB1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/KPNA5","total_profiled":1310},"omim":[{"mim_id":"610563","title":"KARYOPHERIN ALPHA-6; KPNA6","url":"https://www.omim.org/entry/610563"},{"mim_id":"604545","title":"KARYOPHERIN ALPHA-5; KPNA5","url":"https://www.omim.org/entry/604545"},{"mim_id":"603948","title":"NUCLEOPORIN, 153-KD; NUP153","url":"https://www.omim.org/entry/603948"},{"mim_id":"600555","title":"SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1; STAT1","url":"https://www.omim.org/entry/600555"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"testis","ntpm":39.2}],"url":"https://www.proteinatlas.org/search/KPNA5"},"hgnc":{"alias_symbol":["SRP6","IPOA6"],"prev_symbol":[]},"alphafold":{"accession":"O15131","domains":[{"cath_id":"1.25.10.10","chopping":"81-241","consensus_level":"medium","plddt":96.2127,"start":81,"end":241},{"cath_id":"1.25.10.10","chopping":"379-505","consensus_level":"medium","plddt":95.3811,"start":379,"end":505}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O15131","model_url":"https://alphafold.ebi.ac.uk/files/AF-O15131-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O15131-F1-predicted_aligned_error_v6.png","plddt_mean":85.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KPNA5","jax_strain_url":"https://www.jax.org/strain/search?query=KPNA5"},"sequence":{"accession":"O15131","fasta_url":"https://rest.uniprot.org/uniprotkb/O15131.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O15131/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O15131"}},"corpus_meta":[{"pmid":"25121748","id":"PMC_25121748","title":"Ebola 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virology","url":"https://pubmed.ncbi.nlm.nih.gov/7494303","citation_count":245,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10662,"output_tokens":1700,"usd":0.028743},"stage2":{"model":"claude-opus-4-6","input_tokens":4960,"output_tokens":2087,"usd":0.115462},"total_usd":0.247933,"stage1_batch_id":"msgbatch_01Mgbec4vM6c9vXht7JVJzss","stage2_batch_id":"msgbatch_01JkuuH5RB2BdJNsgnhjbC6s","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":22277,"output_tokens":3305,"usd":0.058203},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":6065,"output_tokens":1,"usd":0.045525}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of human KPNA5 C-terminus in complex with Ebola virus VP24 (eVP24) revealed that eVP24 recognizes a unique nonclassical NLS binding site on KPNA5 that is necessary for efficient PY-STAT1 nuclear transport; eVP24 binds KPNA5 with very high affinity to competitively inhibit PY-STAT1 nuclear transport without affecting classical NLS cargo transport.\",\n      \"method\": \"X-ray crystallography, binding affinity measurements, cell-based nuclear transport assays, mutagenesis\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus functional validation with mutagenesis and transport assays in a highly cited study\",\n      \"pmids\": [\"25121748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"KPNA5 (along with KPNA1 and KPNA6) directly interacts with PHB2 (Prohibitin 2) and mediates estrogen-dependent nuclear translocation of PHB2 in breast cancer cells; siRNA knockdown of KPNA5 inhibited E2-dependent PHB2 nuclear translocation, enhancing ERα activation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, nuclear translocation assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — reciprocal Co-IP and functional knockdown with defined phenotype, single lab\",\n      \"pmids\": [\"26052702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Lloviu virus VP24 blocks tyrosine-phosphorylated STAT1 binding to KPNA5, preventing STAT1 nuclear accumulation and IFN-induced gene expression, paralleling EBOV VP24 function.\",\n      \"method\": \"Co-immunoprecipitation, nuclear accumulation assays, reporter gene assays\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional binding and nuclear import assays, single lab\",\n      \"pmids\": [\"26255028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Quantitative comparison of VP24 proteins from EBOV, BDBV, and RESTV binding to KPNA1, KPNA5, and KPNA6 showed that BDBV VP24 has ~10-fold lower KPNA binding affinity; VP24-KPNA interaction affects VP24 protein stability and IFN inhibition capacity, identifying a novel functional consequence of the VP24-KPNA5 interaction.\",\n      \"method\": \"Purified protein binding assays (quantitative affinity measurements), cell-based IFN inhibition assays, mutagenesis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — quantitative in vitro binding with purified proteins, mutagenesis, and cell-based validation\",\n      \"pmids\": [\"27974555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KPNA5 recognizes nuclear localization signals (NLSs) in PTPN4 and mediates PTPN4 nuclear transport, thereby inhibiting STAT3 phosphorylation and downstream signaling in ovarian cancer cells; KPNA5 overexpression suppressed OC cell proliferation, tumor growth, and invasion in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and knockdown experiments, xenograft model, Western blot\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — protein interaction established by Co-IP with defined functional phenotype in vitro and in vivo, single lab\",\n      \"pmids\": [\"40145330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"miR-153 directly targets KPNA5 via two binding sites in its 3'UTR to suppress KPNA5 expression; KPNA5 binds three nuclear localization sequences of NRF2 and facilitates NRF2 nuclear translocation; loss of KPNA5 impairs NRF2-mediated antioxidant responses and promotes oxidative stress in the context of chronic cerebral hypoperfusion.\",\n      \"method\": \"3'UTR reporter assays (miR-153 targeting), co-immunoprecipitation (KPNA5-NRF2 interaction), knockdown experiments in rat model, nuclear translocation assays\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding established with protein interaction assays, miRNA targeting validated with reporters, functional readouts in vivo\",\n      \"pmids\": [\"40816766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Trafficking of eVP24 into the nucleus via KPNA5 requires simultaneous binding of cargo; eVP24 binding to KPNA5 does not impact cargo NLS binding, indicating simultaneous binding of both cellular cargo and eVP24 to KPNA5 is required for nuclear trafficking of VP24.\",\n      \"method\": \"Biophysical assays (conformational dynamics analysis), binding assays with purified proteins, nuclear transport assays\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — biophysical characterization with purified proteins establishing mechanistic model, single lab\",\n      \"pmids\": [\"40872766\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KPNA5 is a nuclear importin (NPI-1 subfamily karyopherin alpha) that mediates nuclear transport of cargo bearing nonclassical NLS sequences, including phosphorylated STAT1, NRF2 (via three NLS sites), and PTPN4; Ebola virus VP24 selectively hijacks a unique nonclassical NLS binding site on KPNA5 to competitively block PY-STAT1 nuclear import and suppress innate immune signaling, while itself being trafficked into the nucleus via KPNA5 through a simultaneous cargo-binding mechanism.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries from KEPT papers only.\n\n**Classification:**\n\nRetrieved papers:\n1. PMID:25121748 — KEEP (KPNA5 structure with eVP24, direct mechanism)\n2. PMID:17889653 — EXCLUDE (C. elegans serpin/necrosis, no KPNA5)\n3. PMID:22302350 — EXCLUDE (somatic mutations survey, no mechanistic data)\n4. PMID:26255028 — KEEP (LLOV VP24 blocks PY-STAT1 binding to KPNA5)\n5. PMID:28574091 — KEEP (macrocyclic peptide inhibitors of VP24-KPNA5 PPI)\n6. PMID:27974555 — KEEP (VP24-KPNA binding affinities, IFN inhibition, VP24 stability)\n7. PMID:27009368 — KEEP (VP24 SDPs affecting KPNA5 binding)\n8. PMID:35022507 — EXCLUDE (lysoptosis/serpin, no KPNA5)\n9. PMID:26052702 — KEEP (BIG3 blocks KPNAs-mediated PHB2 nuclear translocation)\n10. PMID:33553736 — EXCLUDE (expression/prognostic only)\n11. PMID:37259332 — KEEP (miR-944/SNHG6/KPNA5 axis in GEM resistance)\n12. PMID:34696514 — EXCLUDE (expression profiling, no direct mechanism)\n13. PMID:28418440 — KEEP (MD simulation of VP24-KPNA5 binding mechanisms)\n14. PMID:28812539 — KEEP (molecular dynamics VP24-KPNA5 interaction)\n15. PMID:29140409 — KEEP (computational framework VP24-KPNA5 interactions)\n16. PMID:12584438 — EXCLUDE (chromosomal mapping, no mechanistic data)\n17. PMID:40816766 — KEEP (miR-153 targets KPNA5, NRF2 nuclear translocation)\n18. PMID:40145330 — KEEP (KPNA5 imports PTPN4 into nucleus, inhibits STAT3)\n19. PMID:40053206 — KEEP (miR-153-3p targets KPNA5 in macrophage polarization)\n20. PMID:40872766 — KEEP (biophysical basis of eVP24-KPNA5, simultaneous cargo binding)\n\nGene2pubmed curated papers: Most are large interactome/proteomics surveys or HIV matrix/Vpr nuclear import papers not directly about KPNA5 mechanism. PMID:9275210 mentions importin/karyopherin pathway for HIV integrase but not specifically KPNA5. PMID:9463369 mentions karyopherin alpha binding to Vpr but not specifically KPNA5. All gene2pubmed papers are EXCLUDE for direct KPNA5 mechanistic findings (they are either large-scale screens where KPNA5 appears incidentally, or HIV papers about other proteins).\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of the human KPNA5 C-terminus in complex with Ebola virus VP24 (eVP24) revealed that eVP24 recognizes a unique nonclassical NLS binding site on KPNA5 that is necessary for efficient PY-STAT1 nuclear transport. eVP24 binds KPNA5 with very high affinity at this site to competitively inhibit PY-STAT1 nuclear import without affecting classical NLS cargo transport.\",\n      \"method\": \"X-ray crystallography of KPNA5 C-terminus/eVP24 complex; binding affinity measurements; cell-based nuclear transport assays\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus functional validation with mutagenesis and transport assays in a highly cited study\",\n      \"pmids\": [\"25121748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Lloviu virus VP24 (LLOV VP24) blocks tyrosine-phosphorylated STAT1 (PY-STAT1) binding to KPNA5, preventing STAT1 nuclear accumulation and IFN-induced gene expression, demonstrating that KPNA5's role in PY-STAT1 nuclear import is targeted by multiple filovirus VP24 proteins.\",\n      \"method\": \"Co-immunoprecipitation; nuclear accumulation assays; IFN-induced gene expression assays in human and bat cells\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal functional assays in multiple cell types, single lab\",\n      \"pmids\": [\"26255028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PHB2 interacts with KPNA1, KPNA5, and KPNA6, and these interactions mediate estrogen-dependent translocation of PHB2 into the nuclei of breast cancer cells. BIG3 blocks the KPNA-binding region(s) of PHB2, thereby inhibiting KPNA-mediated PHB2 nuclear translocation.\",\n      \"method\": \"Co-immunoprecipitation; siRNA knockdown of individual KPNAs; nuclear translocation assays in breast cancer cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus siRNA knockdown with translocation readout, single lab\",\n      \"pmids\": [\"26052702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"VP24 proteins from different Ebolavirus species (EBOV, BDBV, RESTV) all bind KPNA1, KPNA5, and KPNA6, but with differing affinities; BDBV VP24 shows ~10-fold lower KPNA5 binding affinity than EBOV or RESTV VP24. Reduced VP24-KPNA binding affinity decreases IFN inhibition and shortens VP24 half-life, revealing that KPNA interaction stabilizes VP24.\",\n      \"method\": \"Binding affinity measurements with purified proteins; cell-based VP24 half-life assays; IFN-induced gene expression assays; alanine-scanning mutagenesis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — quantitative binding assays with purified proteins plus multiple functional readouts and mutagenesis\",\n      \"pmids\": [\"27974555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Molecular dynamics simulations of the VP24-KPNA5 complex identified key hydrogen bonds (D480-T138, E483-R137, D205-R396) critical for the interaction interface. VP24 residues R137 and T138, and KPNA5 residues R396, R398, R480, Y477, and F484, were identified as hotspot residues essential for complex stability.\",\n      \"method\": \"All-atom molecular dynamics simulation; MM-GB/SA energy calculation; computational alanine scanning\",\n      \"journal\": \"Molecular bioSystems\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational prediction only, no experimental validation\",\n      \"pmids\": [\"28418440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Macrocyclic peptide inhibitors of the VP24-KPNA5 protein-protein interaction were identified with dissociation constants in the single-digit nanomolar range for eVP24, and these peptides successfully disrupted the eVP24-KPNA interaction, providing the first chemical probes capable of modulating this PPI.\",\n      \"method\": \"RaPID (Random non-standard Peptides Integrated Discovery) system; binding affinity measurements; PPI disruption assays with purified proteins\",\n      \"journal\": \"Organic & biomolecular chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro reconstitution of PPI disruption with purified components, single lab\",\n      \"pmids\": [\"28574091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"miR-153 directly targets two binding sites in the 3'UTR of KPNA5 mRNA to suppress KPNA5 expression. KPNA5 facilitates nuclear translocation of NRF2 by binding to three nuclear localization sequences (NLS) of NRF2, and miR-153-mediated suppression of KPNA5 impairs NRF2-mediated antioxidant responses, promoting oxidative stress and cognitive deficits in chronic cerebral hypoperfusion models.\",\n      \"method\": \"Luciferase reporter assays for miR-153 binding sites; Co-IP for KPNA5-NRF2 interaction; knockdown and overexpression in vivo (rat basal forebrain) and in vitro; nuclear fractionation assays\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus in vivo knockdown with defined cognitive/oxidative phenotype, single lab\",\n      \"pmids\": [\"40816766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KPNA5 recognizes nuclear localization signals (NLS) in the protein tyrosine phosphatase PTPN4 and mediates its nuclear transport. Nuclear PTPN4 inhibits STAT3 phosphorylation and its downstream signaling pathway. KPNA5 overexpression suppressed ovarian cancer cell proliferation, tumor growth, and invasion in vitro and in vivo.\",\n      \"method\": \"Co-IP for KPNA5-PTPN4 interaction; Western blot for STAT3 phosphorylation; xenograft tumor model; CCK-8, colony formation, Transwell assays\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional cellular and in vivo assays with defined pathway readout, single lab\",\n      \"pmids\": [\"40145330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"miR-153-3p derived from bone marrow mesenchymal stem cell exosomes targets KPNA5, and this targeting promotes M2 macrophage polarization to facilitate wound healing.\",\n      \"method\": \"Target prediction and validation; co-culture assays; exosome transfer experiments; macrophage polarization assays\",\n      \"journal\": \"Applied biochemistry and biotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, limited mechanistic follow-up on KPNA5 specifically\",\n      \"pmids\": [\"40053206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Nuclear trafficking of eVP24 by KPNA5 requires simultaneous binding of cellular NLS-containing cargo; eVP24 binding to KPNA5 does not impair cargo NLS binding at the major NLS binding site, indicating that eVP24 and classical cargo can simultaneously associate with KPNA5 for co-transport into the nucleus.\",\n      \"method\": \"Hydrogen-deuterium exchange mass spectrometry (HDX-MS); native MS; biophysical binding assays with purified proteins; nuclear localization assays\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal biophysical methods with purified proteins, single lab\",\n      \"pmids\": [\"40872766\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KPNA5 is a nuclear import receptor (NPI-1 subfamily karyopherin alpha) that transports cargo bearing nonclassical NLS sequences—including phosphorylated STAT1 and NRF2—into the nucleus via a unique binding site on its C-terminus; Ebola virus VP24 exploits this site with high affinity to selectively block PY-STAT1 nuclear import while permitting simultaneous binding of classical NLS cargo, and KPNA5 also mediates nuclear import of PTPN4 (suppressing STAT3 signaling) and PHB2 (suppressing ERα signaling), with its activity regulated post-transcriptionally by miR-153 targeting its 3'UTR.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"KPNA5 is a karyopherin-α family importin that mediates nuclear transport of cargoes bearing nonclassical nuclear localization signals, with particular specificity for tyrosine-phosphorylated STAT1, NRF2, PTPN4, and PHB2 [PMID:25121748, PMID:40145330, PMID:40816766, PMID:26052702]. Structural and biophysical studies demonstrate that KPNA5 possesses a unique nonclassical NLS-binding site in its C-terminal domain that is exploited by Ebola virus VP24, which binds with high affinity to competitively block PY-STAT1 nuclear import and thereby suppress interferon signaling, while VP24 itself is simultaneously co-transported into the nucleus along with classical NLS cargo [PMID:25121748, PMID:40872766]. KPNA5-mediated nuclear import of PTPN4 inhibits STAT3 phosphorylation and suppresses ovarian cancer cell proliferation, and KPNA5-dependent NRF2 nuclear translocation activates antioxidant gene expression, a process impaired when miR-153 silences KPNA5 during chronic cerebral hypoperfusion [PMID:40145330, PMID:40816766].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"The crystal structure of KPNA5 with Ebola VP24 revealed how a filoviral protein hijacks a unique nonclassical NLS-binding site on KPNA5 to selectively block PY-STAT1 nuclear import without disrupting classical NLS cargo transport, establishing KPNA5 as an importin with functionally distinct binding surfaces.\",\n      \"evidence\": \"X-ray crystallography of KPNA5 C-terminus–eVP24 complex, mutagenesis, binding affinity measurements, and cell-based nuclear transport assays\",\n      \"pmids\": [\"25121748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full-length KPNA5 structure with simultaneous classical and nonclassical cargoes not resolved\",\n        \"Endogenous cellular cargoes that use the nonclassical NLS site were not systematically identified\",\n        \"Selectivity determinants distinguishing KPNA5 from paralogues KPNA1 and KPNA6 not fully defined\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extending the viral paradigm, Lloviu virus VP24 was shown to block PY-STAT1 binding to KPNA5 analogously to EBOV VP24, and KPNA5 was independently found to mediate estrogen-dependent nuclear translocation of PHB2 in breast cancer cells, broadening the repertoire of KPNA5-specific cargoes.\",\n      \"evidence\": \"Co-immunoprecipitation, nuclear accumulation and reporter assays for Lloviu VP24; Co-IP and siRNA knockdown with nuclear translocation assays for PHB2\",\n      \"pmids\": [\"26255028\", \"26052702\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"PHB2 interaction demonstrated by Co-IP without structural or in vitro reconstitution data\",\n        \"Whether PHB2 uses the same nonclassical NLS site as PY-STAT1 is unknown\",\n        \"Redundancy among KPNA1/KPNA5/KPNA6 for PHB2 transport not resolved\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Quantitative binding studies across filoviral VP24 orthologues revealed that KPNA5 binding affinity determines VP24 protein stability and IFN-inhibitory potency, demonstrating a functional consequence of the VP24–KPNA5 interaction beyond competitive inhibition of STAT1 import.\",\n      \"evidence\": \"Purified protein quantitative binding assays, mutagenesis, and cell-based IFN inhibition assays comparing EBOV, BDBV, and RESTV VP24\",\n      \"pmids\": [\"27974555\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which KPNA5 binding stabilizes VP24 protein is not defined\",\n        \"Whether endogenous cargo binding similarly affects cargo stability was not tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of PTPN4 and NRF2 as direct KPNA5 cargoes linked KPNA5-mediated nuclear import to STAT3 signaling suppression in ovarian cancer and to NRF2-driven antioxidant defense in cerebral hypoperfusion, and miR-153 was established as a physiological negative regulator of KPNA5 expression.\",\n      \"evidence\": \"Co-IP, overexpression/knockdown, xenograft models for PTPN4; 3′UTR reporter assays, Co-IP, and rat model of chronic hypoperfusion for NRF2/miR-153\",\n      \"pmids\": [\"40145330\", \"40816766\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"PTPN4 and NRF2 interactions each demonstrated by single-lab Co-IP without structural characterization\",\n        \"Which NLS-binding surface on KPNA5 is used by PTPN4 and NRF2 is unknown\",\n        \"In vivo significance of miR-153–KPNA5 axis awaits independent replication\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Biophysical analysis showed that eVP24 nuclear trafficking requires simultaneous binding of a classical NLS cargo to KPNA5, indicating that VP24 does not compete with classical cargo but instead co-opts the loaded importin for its own nuclear entry.\",\n      \"evidence\": \"Conformational dynamics analysis and binding assays with purified KPNA5, eVP24, and NLS peptides; nuclear transport assays\",\n      \"pmids\": [\"40872766\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis of simultaneous cargo and VP24 binding not resolved at atomic level\",\n        \"Whether other nonclassical cargoes (PY-STAT1, NRF2) similarly require co-loading is untested\",\n        \"Single-lab biophysical study; independent confirmation needed\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A comprehensive map of endogenous KPNA5-specific cargoes and the structural basis for cargo selectivity versus paralogues KPNA1 and KPNA6 remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No unbiased proteomics screen for KPNA5-specific cargoes has been reported\",\n        \"Full-length KPNA5 structure with simultaneous classical and nonclassical cargoes is lacking\",\n        \"Physiological contexts where KPNA5 is non-redundant with other NPI-1 subfamily members are poorly defined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 4, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1, 4, 5, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 1, 4, 5, 6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 2, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"STAT1\",\n      \"NRF2\",\n      \"PTPN4\",\n      \"PHB2\",\n      \"KPNB1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n"}