{"gene":"PPIL1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2006,"finding":"NMR solution structure of PPIL1 was determined, revealing a cyclophilin-family fold with PPIase activity. GST pulldown and surface plasmon resonance experiments established that PPIL1 stably associates with the N-terminal region of SKIP (residues 59–129) with a dissociation constant of 1.25×10⁻⁷ M. Chemical shift perturbation NMR mapped the SKIP binding interface on PPIL1 to a site distinct from the PPIase active site.","method":"NMR structure determination, GST pulldown, surface plasmon resonance, chemical shift perturbation NMR","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure, in vitro binding assays, and SPR with quantitative Kd in one rigorous study","pmids":["16595688"],"is_preprint":false},{"year":2009,"finding":"The N-terminal region of SKIP (residues 59–129) is intrinsically disordered; upon binding PPIL1 it undergoes a disorder-to-order transition. NMR structure of the PBF·PPIL1 complex showed that a minimal 21-residue SKIP fragment (residues 59–79) binds PPIL1 via electrostatic and hydrophobic interactions through a region distinct from the PPIase active site, leaving the active site open and available for isomerase/chaperone activity on other spliceosomal substrates.","method":"NMR structure determination of complex, NMR dynamics analysis of intrinsic disorder","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure of complex with mutagenesis-level fragment mapping, single rigorous study with multiple orthogonal NMR methods","pmids":["20007319"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of PPIL1 bound to the inhibitor cyclosporine A (CsA) was solved at 1.15 Å resolution (SAD phasing with Cd²⁺). A peptide array identified a minimal 36-residue linear epitope from SKIP (centred on an 8-residue core) sufficient to bind PPIL1 in pulldown assays. Molecular docking using the crystal structure and NMR data produced a model in which a SKIP proline is buried in a hydrophobic pocket of PPIL1, surrounded by hydrogen bonds, consistent with SKIP recruiting PPIL1 into the spliceosome core.","method":"X-ray crystallography (1.15 Å), peptide array, GST pulldown, molecular docking","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure plus biochemical binding mapping in one study","pmids":["20368803"],"is_preprint":false},{"year":2006,"finding":"PPIL1 promotes colon cancer cell growth: siRNA knockdown of PPIL1 in SNUC4/SNUC5 cells retarded proliferation, and overexpression increased colony formation in NIH3T3 and HEK293 cells. Co-immunoprecipitation identified SNW1/SKIP and stathmin as PPIL1-interacting proteins.","method":"siRNA knockdown + growth assay, colony formation assay, co-immunoprecipitation","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — functional KD phenotype plus Co-IP identification of binding partners; single lab","pmids":["16397026"],"is_preprint":false},{"year":2001,"finding":"Yeast two-hybrid screens in Dictyostelium discoideum and Schizosaccharomyces pombe identified cyclophilins (CypE/Cyp2) as the orthologues of human PPIL1; both bind their respective SNW/SKIP proteins at the N-terminal region of SNW in a cyclosporin A-independent manner, establishing the conserved PPIL1–SKIP interaction. CypE was confirmed in vitro to have cyclosporin A-sensitive PPIase activity.","method":"Yeast two-hybrid, in vitro PPIase activity assay","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — two orthogonal organisms screened independently, PPIase activity confirmed in vitro; limited to yeast/Dictyostelium orthologs","pmids":["11690648"],"is_preprint":false},{"year":2020,"finding":"Biallelic loss-of-function mutations in PPIL1 cause pontocerebellar hypoplasia with microcephaly (PCHM) in humans. PPIL1 patient-mutation knockin mice showed neuron-specific apoptosis. Loss of PPIL1 disrupted splicing integrity, predominantly affecting short and high-GC-content introns and genes linked to brain disorders. PPIL1 and PRP17 form an active isomerase–substrate interaction, but isomerase activity per se is not critical for PPIL1's spliceosomal function, establishing a non-enzymatic role for the prolyl isomerase.","method":"Patient genetics, PPIL1 knockin mouse model (neuronal apoptosis readout), splicing analysis (RNA-seq of affected introns), biochemical isomerase-activity mutagenesis","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — human genetics, mouse KI phenotype, transcriptome splicing analysis, and enzymatic mutagenesis combined in one study replicated across 10 families","pmids":["33220177"],"is_preprint":false},{"year":2004,"finding":"PPIL1 is recruited by SKIP into the spliceosome as a foldase/prolyl isomerase, providing a mechanistic link between the SKIP transcriptional coregulator and spliceosome activation; PPIL1 is a component of the 35S U5 snRNP and the 45S activated spliceosome.","method":"Biochemical fractionation/spliceosome purification (review of primary data)","journal":"Cellular and molecular life sciences : CMLS","confidence":"Low","confidence_rationale":"Tier 3 / Weak — review synthesis of earlier data; no new primary experiment described in this abstract","pmids":["15052407"],"is_preprint":false},{"year":2022,"finding":"Intracellular C3b-α'2 (generated downstream of S1PR1 activation) associates with PPIL1 via glutamic acid 156 (E156) and aspartic acid 111 (D111) of C3b-α'2, and this interaction is required for NLRP3/inflammasome induction and tumor metastasis; mutation of these C3b residues to prevent PPIL1 binding attenuated inflammasome activation and reduced lung colonization in mice.","method":"Site-directed mutagenesis of C3b, co-immunoprecipitation, in vivo lung colonization assay, NLRP3 inflammasome readout","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis identifying specific residues required for C3b–PPIL1 interaction and in vivo functional consequence; single lab","pmids":["36476873"],"is_preprint":false},{"year":2025,"finding":"NMR titration experiments showed that proline-rich PxxP motifs within the EWS low-complexity domain (EWSLCD) engage the catalytic face of PPIL1, forming low-affinity 'fuzzy' complexes. This interaction is absent when the RRM and RGG2 domains are included in the EWS construct, indicating that PxxP accessibility is context-dependent. PPIL1 is recruited into EWSLCD phase-separated condensates and alters their condensation properties at low salt concentrations.","method":"NMR titration, phase separation/droplet assay","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — NMR mapping plus phase separation assay; single lab, single study","pmids":["40668764"],"is_preprint":false},{"year":2025,"finding":"PPIL1 knockdown in HCC cell lines suppressed proliferation, migration, and sphere-forming capacity, and reduced tumor growth in xenograft mice. Transcriptome analysis after PPIL1 knockdown identified DAAM2 as a downstream transcriptional target, linking PPIL1 to Wnt/β-catenin signaling activation in liver cancer stem cells.","method":"shRNA knockdown, xenograft mouse model, sphere formation assay, transcriptome analysis","journal":"Cancer genomics & proteomics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — KD phenotype in vitro and in vivo with transcriptomic pathway identification; single lab","pmids":["40883023"],"is_preprint":false}],"current_model":"PPIL1 is a cyclophilin-family peptidyl-prolyl cis/trans isomerase that localizes to the nucleus as a component of the U5 snRNP and activated spliceosome, where it is recruited by the spliceosomal coregulator SKIP via a conserved binding interface distinct from the PPIase active site (leaving the active site free); its PPIase enzymatic activity is dispensable for spliceosomal function, which instead relies on a non-catalytic structural role, and biallelic loss-of-function mutations disrupt splicing integrity of short, GC-rich introns, causing neuron-specific apoptosis and pontocerebellar hypoplasia with microcephaly; outside the spliceosome, PPIL1 is engaged by the intracellular C3b complement fragment downstream of S1PR1 to activate NLRP3 inflammasome-dependent metastasis, interacts with the EWS low-complexity domain via PxxP motifs at its catalytic face, and promotes cancer cell proliferation at least partly through transcriptional upregulation of DAAM2 and Wnt/β-catenin signaling."},"narrative":{"mechanistic_narrative":"PPIL1 is a cyclophilin-family peptidyl-prolyl cis/trans isomerase that functions principally as a non-catalytic structural component of the spliceosome [PMID:16595688, PMID:33220177]. It is recruited into the U5 snRNP and activated spliceosome by the coregulator SKIP/SNW1, whose intrinsically disordered N-terminal region undergoes a disorder-to-order transition and docks onto a surface of PPIL1 distinct from the PPIase active site, leaving the catalytic pocket free [PMID:16595688, PMID:20007319, PMID:20368803]. This recruitment interface is conserved across species [PMID:11690648]. Although PPIL1 forms an isomerase–substrate interaction with the spliceosomal factor PRP17, its enzymatic activity is dispensable for spliceosomal function, which instead depends on a structural role; biallelic loss-of-function mutations disrupt splicing of short, high-GC-content introns and cause neuron-specific apoptosis and pontocerebellar hypoplasia with microcephaly [PMID:33220177]. Beyond splicing, PPIL1 promotes cancer cell proliferation and tumor growth, acting in liver cancer through transcriptional upregulation of DAAM2 and Wnt/β-catenin signaling [PMID:16397026, PMID:40883023], and is engaged by the intracellular C3b complement fragment downstream of S1PR1 to drive NLRP3 inflammasome activation and metastasis [PMID:36476873]. PPIL1 is also recruited via proline-rich PxxP motifs at its catalytic face into EWS low-complexity-domain condensates, altering their phase-separation properties [PMID:40668764].","teleology":[{"year":2001,"claim":"Established that the PPIL1–SKIP interaction is evolutionarily conserved and cyclosporin A-independent, defining SKIP as the canonical partner of this cyclophilin before its human characterization.","evidence":"Yeast two-hybrid screens in Dictyostelium and S. pombe identifying CypE/Cyp2 orthologues, plus in vitro PPIase assay","pmids":["11690648"],"confidence":"Medium","gaps":["Restricted to non-human orthologs","Did not establish where on PPIL1 SKIP binds","No spliceosomal function tested directly"]},{"year":2006,"claim":"Determined the PPIL1 fold and demonstrated that SKIP binds at a site distinct from the PPIase active site, raising the question of whether enzymatic and scaffolding functions are separable.","evidence":"NMR structure, GST pulldown, SPR (Kd = 1.25×10⁻⁷ M), chemical shift perturbation NMR","pmids":["16595688"],"confidence":"High","gaps":["Did not resolve the bound complex structure","Functional consequence of leaving active site free not tested"]},{"year":2006,"claim":"Linked PPIL1 to cancer cell growth and physically confirmed SKIP/SNW1 and stathmin as interactors in human cells.","evidence":"siRNA knockdown growth assay, colony formation, co-immunoprecipitation in colon cancer cells","pmids":["16397026"],"confidence":"Medium","gaps":["Mechanism connecting PPIL1 to proliferation unresolved","Stathmin interaction not followed up mechanistically","Single lab"]},{"year":2009,"claim":"Showed the SKIP N-terminus is intrinsically disordered and folds upon binding PPIL1 through a minimal 21-residue fragment, explaining how recruitment occurs while preserving an open active site.","evidence":"NMR structure of PBF·PPIL1 complex and NMR dynamics analysis","pmids":["20007319"],"confidence":"High","gaps":["Identity of physiological active-site substrates not defined","In-spliceosome geometry not resolved"]},{"year":2010,"claim":"Provided a high-resolution structural and docking model of how SKIP recruits PPIL1 into the spliceosome core via a defined linear epitope.","evidence":"1.15 Å crystal structure of PPIL1·CsA, peptide array epitope mapping, molecular docking","pmids":["20368803"],"confidence":"High","gaps":["Docking model not validated by complex crystal structure","Catalytic role within assembled spliceosome unresolved"]},{"year":2020,"claim":"Resolved the long-standing question of whether PPIL1's isomerase activity is required for function, establishing a non-enzymatic spliceosomal role and a causal link to human disease.","evidence":"Patient genetics across 10 families, knockin mouse with neuronal apoptosis, RNA-seq splicing analysis, isomerase-activity mutagenesis; PRP17 identified as isomerase substrate","pmids":["33220177"],"confidence":"High","gaps":["Why short GC-rich introns are selectively vulnerable not mechanistically explained","Structural basis of the non-catalytic role undefined","Neuron-specificity of phenotype not fully explained"]},{"year":2022,"claim":"Identified a spliceosome-independent role linking PPIL1 to innate immune signaling and metastasis through intracellular complement.","evidence":"Site-directed mutagenesis of C3b (E156/D111), co-IP, in vivo lung colonization, NLRP3 inflammasome readout","pmids":["36476873"],"confidence":"Medium","gaps":["PPIL1 residues mediating C3b binding not mapped","Mechanism connecting PPIL1 to NLRP3 activation unresolved","Single lab"]},{"year":2025,"claim":"Demonstrated that PPIL1 engages proline-rich PxxP motifs of the EWS low-complexity domain at its catalytic face and modulates condensate properties, implicating it in phase separation.","evidence":"NMR titration and phase separation/droplet assays","pmids":["40668764"],"confidence":"Medium","gaps":["Low-affinity fuzzy interaction physiological relevance unclear","Cellular consequence of condensate modulation untested","Context-dependent (blocked by RRM/RGG2 domains)"]},{"year":2025,"claim":"Connected PPIL1's pro-proliferative role to a specific transcriptional output in liver cancer.","evidence":"shRNA knockdown, xenograft model, sphere formation, transcriptome analysis identifying DAAM2/Wnt-β-catenin","pmids":["40883023"],"confidence":"Medium","gaps":["How a spliceosomal isomerase regulates DAAM2 transcription unresolved","Direct vs indirect effect on Wnt signaling unclear","Single lab"]},{"year":null,"claim":"How a single splicing-associated cyclophilin mediates such diverse spliceosome-independent functions (complement/inflammasome, condensate modulation, Wnt-driven proliferation), and what structural feature underlies its catalysis-independent spliceosomal role, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying mechanism linking nuclear splicing and cytoplasmic/immune roles","Physiological substrate(s) of PPIL1 PPIase activity unknown","Structural basis of non-catalytic spliceosomal function undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0,4,5]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,5]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,5]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[5,6]}],"complexes":["U5 snRNP","activated spliceosome"],"partners":["SNW1","PRP17","STMN1","C3","EWSR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y3C6","full_name":"Peptidyl-prolyl cis-trans isomerase-like 1","aliases":["Rotamase PPIL1"],"length_aa":166,"mass_kda":18.2,"function":"Involved in pre-mRNA splicing as component of the spliceosome (PubMed:11991638, PubMed:28076346, PubMed:28502770, PubMed:33220177). PPIases accelerate the folding of proteins. Catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides (PubMed:16595688). Catalyzes prolyl peptide bond isomerization in CDC40/PRP17 (PubMed:33220177). Plays an important role in embryonic brain development; this function is independent of its isomerase activity (PubMed:33220177)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9Y3C6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/PPIL1","classification":"Common Essential","n_dependent_lines":974,"n_total_lines":1208,"dependency_fraction":0.8062913907284768},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CPSF6","stoichiometry":0.2},{"gene":"RBM39","stoichiometry":0.2},{"gene":"SF3A1","stoichiometry":0.2},{"gene":"SF3B1","stoichiometry":0.2},{"gene":"SNRPA","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2},{"gene":"SNRPC","stoichiometry":0.2},{"gene":"SNRPF","stoichiometry":0.2},{"gene":"SSRP1","stoichiometry":0.2},{"gene":"TOP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PPIL1","total_profiled":1310},"omim":[{"mim_id":"619302","title":"PONTOCEREBELLAR HYPOPLASIA, TYPE 15; PCH15","url":"https://www.omim.org/entry/619302"},{"mim_id":"619301","title":"PONTOCEREBELLAR HYPOPLASIA, TYPE 14; PCH14","url":"https://www.omim.org/entry/619301"},{"mim_id":"607596","title":"PONTOCEREBELLAR HYPOPLASIA, TYPE 1A; PCH1A","url":"https://www.omim.org/entry/607596"},{"mim_id":"605585","title":"CELL DIVISION CYCLE 40; CDC40","url":"https://www.omim.org/entry/605585"},{"mim_id":"601301","title":"PEPTIDYL-PROLYL ISOMERASE-LIKE 1; PPIL1","url":"https://www.omim.org/entry/601301"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PPIL1"},"hgnc":{"alias_symbol":["CYPL1"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y3C6","domains":[{"cath_id":"2.40.100.10","chopping":"12-163","consensus_level":"high","plddt":95.3449,"start":12,"end":163}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3C6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3C6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3C6-F1-predicted_aligned_error_v6.png","plddt_mean":94.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPIL1","jax_strain_url":"https://www.jax.org/strain/search?query=PPIL1"},"sequence":{"accession":"Q9Y3C6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y3C6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y3C6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3C6"}},"corpus_meta":[{"pmid":"25033284","id":"PMC_25033284","title":"Integrative genomics reveals novel molecular pathways and gene networks for coronary artery disease.","date":"2014","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/25033284","citation_count":159,"is_preprint":false},{"pmid":"15052407","id":"PMC_15052407","title":"Transcriptional coregulator SNW/SKIP: the concealed tie of dissimilar pathways.","date":"2004","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/15052407","citation_count":71,"is_preprint":false},{"pmid":"33220177","id":"PMC_33220177","title":"Mutations in Spliceosomal Genes PPIL1 and PRP17 Cause Neurodegenerative Pontocerebellar Hypoplasia with Microcephaly.","date":"2020","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/33220177","citation_count":48,"is_preprint":false},{"pmid":"16595688","id":"PMC_16595688","title":"Solution structure of human peptidyl prolyl isomerase-like protein 1 and insights into its interaction with SKIP.","date":"2006","source":"The Journal of biological 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Part A","url":"https://pubmed.ncbi.nlm.nih.gov/37159429","citation_count":1,"is_preprint":false},{"pmid":"40883023","id":"PMC_40883023","title":"PPIL1 Drives Hepatocellular Carcinoma Progression and Cancer Stem Cell Self-renewal Through DAAM2-mediated Wnt/β-Catenin Activation.","date":"2025","source":"Cancer genomics & proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/40883023","citation_count":1,"is_preprint":false},{"pmid":"33476558","id":"PMC_33476558","title":"Splicing Control of Pontocerebellar Development.","date":"2021","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/33476558","citation_count":1,"is_preprint":false},{"pmid":"38955739","id":"PMC_38955739","title":"[The diagnostic value of inflammation-related genes in bronchopulmonary dysplasia].","date":"2024","source":"Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine]","url":"https://pubmed.ncbi.nlm.nih.gov/38955739","citation_count":1,"is_preprint":false},{"pmid":"35236814","id":"PMC_35236814","title":"Nuclear localization of propiece IL-1α in HeLa cells.","date":"2022","source":"Journal of oral science","url":"https://pubmed.ncbi.nlm.nih.gov/35236814","citation_count":1,"is_preprint":false},{"pmid":"41073920","id":"PMC_41073920","title":"Obesity induced by a high-fat diet regulates the MYC‒PPIL1 network in the mediation of asthenozoospermia.","date":"2025","source":"Basic and clinical andrology","url":"https://pubmed.ncbi.nlm.nih.gov/41073920","citation_count":0,"is_preprint":false},{"pmid":"41739400","id":"PMC_41739400","title":"Exploring potential biomarkers of diffuse large B-cell lymphoma through multi-dimensional data.","date":"2026","source":"Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico","url":"https://pubmed.ncbi.nlm.nih.gov/41739400","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12015,"output_tokens":2816,"usd":0.039142,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10125,"output_tokens":3148,"usd":0.064662,"stage2_stop_reason":"end_turn"},"total_usd":0.103804,"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\": 2006,\n      \"finding\": \"NMR solution structure of PPIL1 was determined, revealing a cyclophilin-family fold with PPIase activity. GST pulldown and surface plasmon resonance experiments established that PPIL1 stably associates with the N-terminal region of SKIP (residues 59–129) with a dissociation constant of 1.25×10⁻⁷ M. Chemical shift perturbation NMR mapped the SKIP binding interface on PPIL1 to a site distinct from the PPIase active site.\",\n      \"method\": \"NMR structure determination, GST pulldown, surface plasmon resonance, chemical shift perturbation NMR\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure, in vitro binding assays, and SPR with quantitative Kd in one rigorous study\",\n      \"pmids\": [\"16595688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The N-terminal region of SKIP (residues 59–129) is intrinsically disordered; upon binding PPIL1 it undergoes a disorder-to-order transition. NMR structure of the PBF·PPIL1 complex showed that a minimal 21-residue SKIP fragment (residues 59–79) binds PPIL1 via electrostatic and hydrophobic interactions through a region distinct from the PPIase active site, leaving the active site open and available for isomerase/chaperone activity on other spliceosomal substrates.\",\n      \"method\": \"NMR structure determination of complex, NMR dynamics analysis of intrinsic disorder\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure of complex with mutagenesis-level fragment mapping, single rigorous study with multiple orthogonal NMR methods\",\n      \"pmids\": [\"20007319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of PPIL1 bound to the inhibitor cyclosporine A (CsA) was solved at 1.15 Å resolution (SAD phasing with Cd²⁺). A peptide array identified a minimal 36-residue linear epitope from SKIP (centred on an 8-residue core) sufficient to bind PPIL1 in pulldown assays. Molecular docking using the crystal structure and NMR data produced a model in which a SKIP proline is buried in a hydrophobic pocket of PPIL1, surrounded by hydrogen bonds, consistent with SKIP recruiting PPIL1 into the spliceosome core.\",\n      \"method\": \"X-ray crystallography (1.15 Å), peptide array, GST pulldown, molecular docking\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure plus biochemical binding mapping in one study\",\n      \"pmids\": [\"20368803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PPIL1 promotes colon cancer cell growth: siRNA knockdown of PPIL1 in SNUC4/SNUC5 cells retarded proliferation, and overexpression increased colony formation in NIH3T3 and HEK293 cells. Co-immunoprecipitation identified SNW1/SKIP and stathmin as PPIL1-interacting proteins.\",\n      \"method\": \"siRNA knockdown + growth assay, colony formation assay, co-immunoprecipitation\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — functional KD phenotype plus Co-IP identification of binding partners; single lab\",\n      \"pmids\": [\"16397026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Yeast two-hybrid screens in Dictyostelium discoideum and Schizosaccharomyces pombe identified cyclophilins (CypE/Cyp2) as the orthologues of human PPIL1; both bind their respective SNW/SKIP proteins at the N-terminal region of SNW in a cyclosporin A-independent manner, establishing the conserved PPIL1–SKIP interaction. CypE was confirmed in vitro to have cyclosporin A-sensitive PPIase activity.\",\n      \"method\": \"Yeast two-hybrid, in vitro PPIase activity assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — two orthogonal organisms screened independently, PPIase activity confirmed in vitro; limited to yeast/Dictyostelium orthologs\",\n      \"pmids\": [\"11690648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Biallelic loss-of-function mutations in PPIL1 cause pontocerebellar hypoplasia with microcephaly (PCHM) in humans. PPIL1 patient-mutation knockin mice showed neuron-specific apoptosis. Loss of PPIL1 disrupted splicing integrity, predominantly affecting short and high-GC-content introns and genes linked to brain disorders. PPIL1 and PRP17 form an active isomerase–substrate interaction, but isomerase activity per se is not critical for PPIL1's spliceosomal function, establishing a non-enzymatic role for the prolyl isomerase.\",\n      \"method\": \"Patient genetics, PPIL1 knockin mouse model (neuronal apoptosis readout), splicing analysis (RNA-seq of affected introns), biochemical isomerase-activity mutagenesis\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — human genetics, mouse KI phenotype, transcriptome splicing analysis, and enzymatic mutagenesis combined in one study replicated across 10 families\",\n      \"pmids\": [\"33220177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PPIL1 is recruited by SKIP into the spliceosome as a foldase/prolyl isomerase, providing a mechanistic link between the SKIP transcriptional coregulator and spliceosome activation; PPIL1 is a component of the 35S U5 snRNP and the 45S activated spliceosome.\",\n      \"method\": \"Biochemical fractionation/spliceosome purification (review of primary data)\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — review synthesis of earlier data; no new primary experiment described in this abstract\",\n      \"pmids\": [\"15052407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Intracellular C3b-α'2 (generated downstream of S1PR1 activation) associates with PPIL1 via glutamic acid 156 (E156) and aspartic acid 111 (D111) of C3b-α'2, and this interaction is required for NLRP3/inflammasome induction and tumor metastasis; mutation of these C3b residues to prevent PPIL1 binding attenuated inflammasome activation and reduced lung colonization in mice.\",\n      \"method\": \"Site-directed mutagenesis of C3b, co-immunoprecipitation, in vivo lung colonization assay, NLRP3 inflammasome readout\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis identifying specific residues required for C3b–PPIL1 interaction and in vivo functional consequence; single lab\",\n      \"pmids\": [\"36476873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NMR titration experiments showed that proline-rich PxxP motifs within the EWS low-complexity domain (EWSLCD) engage the catalytic face of PPIL1, forming low-affinity 'fuzzy' complexes. This interaction is absent when the RRM and RGG2 domains are included in the EWS construct, indicating that PxxP accessibility is context-dependent. PPIL1 is recruited into EWSLCD phase-separated condensates and alters their condensation properties at low salt concentrations.\",\n      \"method\": \"NMR titration, phase separation/droplet assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — NMR mapping plus phase separation assay; single lab, single study\",\n      \"pmids\": [\"40668764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PPIL1 knockdown in HCC cell lines suppressed proliferation, migration, and sphere-forming capacity, and reduced tumor growth in xenograft mice. Transcriptome analysis after PPIL1 knockdown identified DAAM2 as a downstream transcriptional target, linking PPIL1 to Wnt/β-catenin signaling activation in liver cancer stem cells.\",\n      \"method\": \"shRNA knockdown, xenograft mouse model, sphere formation assay, transcriptome analysis\",\n      \"journal\": \"Cancer genomics & proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — KD phenotype in vitro and in vivo with transcriptomic pathway identification; single lab\",\n      \"pmids\": [\"40883023\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPIL1 is a cyclophilin-family peptidyl-prolyl cis/trans isomerase that localizes to the nucleus as a component of the U5 snRNP and activated spliceosome, where it is recruited by the spliceosomal coregulator SKIP via a conserved binding interface distinct from the PPIase active site (leaving the active site free); its PPIase enzymatic activity is dispensable for spliceosomal function, which instead relies on a non-catalytic structural role, and biallelic loss-of-function mutations disrupt splicing integrity of short, GC-rich introns, causing neuron-specific apoptosis and pontocerebellar hypoplasia with microcephaly; outside the spliceosome, PPIL1 is engaged by the intracellular C3b complement fragment downstream of S1PR1 to activate NLRP3 inflammasome-dependent metastasis, interacts with the EWS low-complexity domain via PxxP motifs at its catalytic face, and promotes cancer cell proliferation at least partly through transcriptional upregulation of DAAM2 and Wnt/β-catenin signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPIL1 is a cyclophilin-family peptidyl-prolyl cis/trans isomerase that functions principally as a non-catalytic structural component of the spliceosome [#0, #5]. It is recruited into the U5 snRNP and activated spliceosome by the coregulator SKIP/SNW1, whose intrinsically disordered N-terminal region undergoes a disorder-to-order transition and docks onto a surface of PPIL1 distinct from the PPIase active site, leaving the catalytic pocket free [#0, #1, #2]. This recruitment interface is conserved across species [#4]. Although PPIL1 forms an isomerase–substrate interaction with the spliceosomal factor PRP17, its enzymatic activity is dispensable for spliceosomal function, which instead depends on a structural role; biallelic loss-of-function mutations disrupt splicing of short, high-GC-content introns and cause neuron-specific apoptosis and pontocerebellar hypoplasia with microcephaly [#5]. Beyond splicing, PPIL1 promotes cancer cell proliferation and tumor growth, acting in liver cancer through transcriptional upregulation of DAAM2 and Wnt/β-catenin signaling [#3, #9], and is engaged by the intracellular C3b complement fragment downstream of S1PR1 to drive NLRP3 inflammasome activation and metastasis [#7]. PPIL1 is also recruited via proline-rich PxxP motifs at its catalytic face into EWS low-complexity-domain condensates, altering their phase-separation properties [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established that the PPIL1–SKIP interaction is evolutionarily conserved and cyclosporin A-independent, defining SKIP as the canonical partner of this cyclophilin before its human characterization.\",\n      \"evidence\": \"Yeast two-hybrid screens in Dictyostelium and S. pombe identifying CypE/Cyp2 orthologues, plus in vitro PPIase assay\",\n      \"pmids\": [\"11690648\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Restricted to non-human orthologs\", \"Did not establish where on PPIL1 SKIP binds\", \"No spliceosomal function tested directly\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Determined the PPIL1 fold and demonstrated that SKIP binds at a site distinct from the PPIase active site, raising the question of whether enzymatic and scaffolding functions are separable.\",\n      \"evidence\": \"NMR structure, GST pulldown, SPR (Kd = 1.25×10⁻⁷ M), chemical shift perturbation NMR\",\n      \"pmids\": [\"16595688\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not resolve the bound complex structure\", \"Functional consequence of leaving active site free not tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Linked PPIL1 to cancer cell growth and physically confirmed SKIP/SNW1 and stathmin as interactors in human cells.\",\n      \"evidence\": \"siRNA knockdown growth assay, colony formation, co-immunoprecipitation in colon cancer cells\",\n      \"pmids\": [\"16397026\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism connecting PPIL1 to proliferation unresolved\", \"Stathmin interaction not followed up mechanistically\", \"Single lab\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed the SKIP N-terminus is intrinsically disordered and folds upon binding PPIL1 through a minimal 21-residue fragment, explaining how recruitment occurs while preserving an open active site.\",\n      \"evidence\": \"NMR structure of PBF·PPIL1 complex and NMR dynamics analysis\",\n      \"pmids\": [\"20007319\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Identity of physiological active-site substrates not defined\", \"In-spliceosome geometry not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided a high-resolution structural and docking model of how SKIP recruits PPIL1 into the spliceosome core via a defined linear epitope.\",\n      \"evidence\": \"1.15 Å crystal structure of PPIL1·CsA, peptide array epitope mapping, molecular docking\",\n      \"pmids\": [\"20368803\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Docking model not validated by complex crystal structure\", \"Catalytic role within assembled spliceosome unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved the long-standing question of whether PPIL1's isomerase activity is required for function, establishing a non-enzymatic spliceosomal role and a causal link to human disease.\",\n      \"evidence\": \"Patient genetics across 10 families, knockin mouse with neuronal apoptosis, RNA-seq splicing analysis, isomerase-activity mutagenesis; PRP17 identified as isomerase substrate\",\n      \"pmids\": [\"33220177\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Why short GC-rich introns are selectively vulnerable not mechanistically explained\", \"Structural basis of the non-catalytic role undefined\", \"Neuron-specificity of phenotype not fully explained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified a spliceosome-independent role linking PPIL1 to innate immune signaling and metastasis through intracellular complement.\",\n      \"evidence\": \"Site-directed mutagenesis of C3b (E156/D111), co-IP, in vivo lung colonization, NLRP3 inflammasome readout\",\n      \"pmids\": [\"36476873\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"PPIL1 residues mediating C3b binding not mapped\", \"Mechanism connecting PPIL1 to NLRP3 activation unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that PPIL1 engages proline-rich PxxP motifs of the EWS low-complexity domain at its catalytic face and modulates condensate properties, implicating it in phase separation.\",\n      \"evidence\": \"NMR titration and phase separation/droplet assays\",\n      \"pmids\": [\"40668764\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Low-affinity fuzzy interaction physiological relevance unclear\", \"Cellular consequence of condensate modulation untested\", \"Context-dependent (blocked by RRM/RGG2 domains)\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected PPIL1's pro-proliferative role to a specific transcriptional output in liver cancer.\",\n      \"evidence\": \"shRNA knockdown, xenograft model, sphere formation, transcriptome analysis identifying DAAM2/Wnt-β-catenin\",\n      \"pmids\": [\"40883023\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"How a spliceosomal isomerase regulates DAAM2 transcription unresolved\", \"Direct vs indirect effect on Wnt signaling unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single splicing-associated cyclophilin mediates such diverse spliceosome-independent functions (complement/inflammasome, condensate modulation, Wnt-driven proliferation), and what structural feature underlies its catalysis-independent spliceosomal role, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No unifying mechanism linking nuclear splicing and cytoplasmic/immune roles\", \"Physiological substrate(s) of PPIL1 PPIase activity unknown\", \"Structural basis of non-catalytic spliceosomal function undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"complexes\": [\"U5 snRNP\", \"activated spliceosome\"],\n    \"partners\": [\"SNW1\", \"PRP17\", \"STMN1\", \"C3\", \"EWSR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}