{"gene":"CNN1","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1988,"finding":"Calponin was purified from bovine aortic smooth muscle as a novel troponin T-like protein that binds actin, tropomyosin, and calmodulin. It was identified as a component of the smooth muscle thin filament with potential regulatory roles in the contractile apparatus.","method":"Protein purification, SDS-PAGE, isoelectric focusing, calmodulin-binding assay, tropomyosin-binding assay, immunological cross-reactivity with troponin T","journal":"Hypertension","confidence":"High","confidence_rationale":"Tier 1 — original biochemical purification and characterization with multiple binding assays; foundational discovery paper","pmids":["2455687"],"is_preprint":false},{"year":1990,"finding":"Calponin (CNN1) inhibits actin-activated myosin MgATPase activity in a reconstituted smooth muscle contractile system. Inhibition requires calponin–actin interaction (not calponin–tropomyosin interaction) and is independent of Ca2+ or myosin phosphorylation state. Phosphorylation of calponin by protein kinase C or Ca2+/calmodulin-dependent protein kinase II abolishes ATPase inhibition and greatly reduces actin binding, while calmodulin- and tropomyosin-binding capabilities are retained.","method":"Reconstituted actomyosin ATPase assay, in vitro phosphorylation by PKC and CaM-kinase II, actin-binding assay, calmodulin-binding assay, tropomyosin-binding assay","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mechanistic dissection of actin-binding requirement; foundational mechanism paper replicated across field","pmids":["2161834"],"is_preprint":false},{"year":1995,"finding":"Human basic calponin (CNN1) cDNA was cloned from human aorta, encoding 297 amino acids (33,169 Da). CNN1 mRNA is expressed exclusively in smooth muscle tissues. The CNN1 gene localizes to chromosome 19p13.1–13.2 by fluorescent in situ hybridization.","method":"cDNA cloning, RT-PCR tissue expression profiling, fluorescent in situ hybridization (FISH)","journal":"Biochemical and Biophysical Research Communications","confidence":"High","confidence_rationale":"Tier 1 — direct molecular cloning and chromosomal mapping","pmids":["8526917"],"is_preprint":false},{"year":1997,"finding":"In differentiated vascular smooth muscle cells, calponin co-immunoprecipitates with mitogen-activated protein kinase (MAPK) and with protein kinase C-epsilon (PKC-ε). An overlay assay confirmed direct interaction between MAPK and calponin, though MAPK does not phosphorylate calponin in vitro. During phenylephrine stimulation, calponin redistributes to the cell membrane coincident with MAPK and PKC-ε membrane targeting, suggesting calponin acts as an adapter/scaffold linking these kinases to the plasma membrane.","method":"Co-immunoprecipitation, overlay assay, in vitro kinase assay, high-resolution digital confocal microscopy","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP and overlay with functional imaging; single lab study","pmids":["9312127"],"is_preprint":false},{"year":2003,"finding":"Calponin H1 (CNN1) is a component of a cGMP kinase signaling complex in tracheal smooth muscle membranes, co-immunoprecipitating with IP3 receptor I, IRAG, cGMP kinase I, alpha-actin, and phospholamban. The complex was validated by mutual co-immunoprecipitation using antibodies to each member protein.","method":"Protein complex purification, MALDI-TOF mass spectrometry, MS/MS sequencing, immunoprecipitation with multiple antibodies, Western blotting","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal co-IP across multiple antibodies identifying complex membership; single lab","pmids":["12480535"],"is_preprint":false},{"year":2011,"finding":"CNN1 overexpression suppresses neointimal formation following arterial injury in transgenic mice. A single intronic CArG element (serum response factor binding site) is necessary but insufficient for correct smooth muscle cell-restricted expression of CNN1 in vivo; deletion of orthologous intronic sequences abolishes endogenous Cnn1 promoter activity.","method":"Transgenic mouse model (BAC transgenics), BAC recombineering with CArG element mutations, femoral artery injury model, immunohistochemistry, Western blotting, lacZ reporter assay","journal":"Arteriosclerosis, Thrombosis, and Vascular Biology","confidence":"High","confidence_rationale":"Tier 2 — in vivo gain-of-function with defined phenotypic readout, CArG mutation series confirms regulatory element requirement","pmids":["21817093"],"is_preprint":false},{"year":2015,"finding":"XBP1s (spliced XBP1) decreases CNN1 expression in smooth muscle cells by directing transcription of miR-1274B, which targets CNN1 mRNA for degradation, thereby promoting SMC proliferation. TGF-β3 (but not TGF-β1 or TGF-β2) attenuates XBP1s-induced CNN1 decrease and SMC proliferation. XBP1 deficiency in SMCs abrogates neointimal formation in vivo.","method":"SMC knockdown/overexpression, miRNA target validation, femoral artery injury mouse model, proteomic analysis of culture media, RT-PCR, Western blotting","journal":"Arteriosclerosis, Thrombosis, and Vascular Biology","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo model with mechanistic dissection of miRNA-mediated CNN1 regulation; single lab","pmids":["26315405"],"is_preprint":false},{"year":2016,"finding":"CNN1 (calponin 1) is specifically expressed in smooth muscle cells and functions to inhibit actin-activated myosin ATPase and stabilize the actin cytoskeleton, fine-tuning smooth muscle contractility. Phosphorylation regulates CNN1 function. The CNN1 gene is one of three vertebrate calponin isoform genes, with smooth muscle-restricted expression.","method":"Review synthesizing in vitro ATPase assays, phosphorylation studies, expression profiling, and cytoskeletal analyses from multiple studies","journal":"Gene","confidence":"High","confidence_rationale":"Tier 1 — review consolidating decades of replicated biochemical and cell biological data","pmids":["26970176"],"is_preprint":false},{"year":2020,"finding":"miR-106b-5p directly binds to the CNN1 mRNA 3'UTR (validated by dual-luciferase assay) and suppresses CNN1 expression, activating the Rho/ROCK1 signaling pathway to promote breast cancer cell survival, migration, invasion, adhesion, and in vivo lung metastasis.","method":"Dual-luciferase reporter assay, CNN1 overexpression/knockdown in breast cancer cell lines, cell viability/migration/invasion/adhesion assays, in vivo lung metastasis assay, transcript half-life assay, Rho/ROCK1 inhibition assay","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2 — direct 3'UTR binding validated, pathway inhibition rescue; single lab","pmids":["31986487"],"is_preprint":false},{"year":2020,"finding":"LINC00337 recruits DNMT1 to the promoter region of CNN1, causing hypermethylation and transcriptional silencing of CNN1 in colorectal cancer. Reduced CNN1 expression promotes CRC cell proliferation, migration/invasion, and tumor angiogenesis (VEGF upregulation), whereas CNN1 overexpression suppresses these phenotypes in vitro and in vivo.","method":"Bisulfite sequencing PCR (BSP), methylation-specific PCR (MSP), loss/gain-of-function experiments, MTT assay, Transwell assay, tube formation assay, nude mouse xenograft, DNMT1 recruitment ChIP-like assay","journal":"Cancer Gene Therapy","confidence":"Medium","confidence_rationale":"Tier 2-3 — epigenetic mechanism validated by BSP/MSP with functional rescue; single lab","pmids":["33328585"],"is_preprint":false},{"year":2021,"finding":"CNN1 overexpression in lung squamous cell carcinoma cells activates TIMP2 expression and inhibits the DKK1/Wnt/β-catenin/c-myc signaling pathway, suppressing invasion, migration, and epithelial-to-mesenchymal transition (EMT). Inhibition of DKK1 reverses these CNN1-mediated effects, restoring β-catenin and c-myc expression.","method":"CNN1 transient overexpression, GEPIA database correlation analysis, wound healing assay, Transwell invasion assay, RT-qPCR, Western blotting, immunofluorescence staining","journal":"Experimental and Therapeutic Medicine","confidence":"Low","confidence_rationale":"Tier 3 — overexpression with pathway marker readout but no direct biochemical interaction; single lab","pmids":["34178128"],"is_preprint":false},{"year":2022,"finding":"CNN1 in cancer-associated fibroblasts (CAFs) interacts with PDZ and LIM Domain 7 (PDLIM7) and prevents its degradation by the E3 ubiquitin ligase NEDD4-1, stabilizing PDLIM7 and thereby activating the ROCK1/MLC pathway. This increases matrix stiffness (CAF contraction), which in turn activates YAP in adjacent gastric cancer cells to confer 5-fluorouracil resistance.","method":"Co-immunoprecipitation, CAF knockdown in 3D culture and mouse GC model, atomic force microscopy for matrix stiffness, NEDD4-1 ubiquitination assay, YAP activity readout","journal":"Matrix Biology","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP identifies binding partner, ubiquitination assay defines mechanism, in vivo rescue; single lab","pmids":["36423735"],"is_preprint":false},{"year":2022,"finding":"CNN1 overexpression in bladder cancer cells suppresses glycolysis by decreasing protein levels of GLUT1, PKM2, and LDHA, with concomitant reduction of HIF-1α and PDK1 mRNA and protein levels, inhibiting tumor cell proliferation, invasion, and migration in vitro and in vivo.","method":"CNN1 overexpression in BC cell lines, CCK8 proliferation assay, Transwell assay, Western blotting, RT-PCR, glucose uptake/lactate secretion/ATP level kits, nude mouse xenograft, immunohistochemistry","journal":"Frontiers in Oncology","confidence":"Low","confidence_rationale":"Tier 3 — overexpression with pathway marker readout; no direct biochemical interaction demonstrated; single lab","pmids":["35903683"],"is_preprint":false},{"year":2024,"finding":"UTX (Kdm6a/lysine demethylase 6A) forms an epigenetic regulatory axis by binding to CNN1, orchestrating trauma-induced senescence of spinal cord microvascular endothelial cells (SCMECs) and their senescence-associated secretory phenotype (SASP). UTX upregulation after spinal cord injury induces CNN1-dependent SCMEC senescence and proinflammatory SASP release; UTX deletion protects SCMECs against senescence and promotes neurological recovery.","method":"Spinal cord injury mouse model, endothelial cell-specific UTX deletion, UTX overexpression, binding assay (UTX–CNN1 interaction), senescence markers, SASP cytokine profiling, functional neurological recovery assessment, senolytic drug delivery","journal":"Bone Research","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo genetic model with defined molecular interaction and functional phenotype; single lab","pmids":["38528029"],"is_preprint":false},{"year":2024,"finding":"lncRNA MEG3 acts as a ceRNA (competing endogenous RNA) by directly binding miR-330, preventing miR-330 from suppressing CNN1 expression in breast cancer. Overexpression of MEG3 inhibits breast cancer cell viability, migration, invasion, and promotes apoptosis; these effects are reversed by miR-330 mimic, which decreases CNN1 levels. Validated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP).","method":"Dual-luciferase reporter assay, RNA immunoprecipitation (RIP), CNN1/MEG3 overexpression/knockdown, cell viability, migration/invasion assays, flow cytometry for apoptosis and cell cycle","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2 — direct RNA–RNA interaction validated by RIP and luciferase; functional rescue experiments; single lab","pmids":["38240701"],"is_preprint":false},{"year":2024,"finding":"CNN1 knockdown by siRNA in chicken (Ross 708 and Randombred) breast muscle satellite cells reduces proliferation, affects differentiation, and decreases lipid accumulation during differentiation, demonstrating a functional role for CNN1 in satellite cell-mediated muscle growth and regeneration.","method":"siRNA knockdown of CNN1, satellite cell isolation, proliferation assay, differentiation assay, lipid accumulation assay","journal":"Poultry Science","confidence":"Low","confidence_rationale":"Tier 3 — clean knockdown with defined phenotype but no pathway placement; single lab, avian model","pmids":["38669818"],"is_preprint":false},{"year":2025,"finding":"USP48 (ubiquitin-specific protease 48) stabilizes and upregulates CNN1 protein in cardiomyocytes; CNN1 silencing abrogates the cardioprotective effects of USP48. The USP48/CNN1 axis inhibits release of CXCL1 and CXCL2 by inactivating the ERK1/2 pathway, protecting against diabetes-aggravated myocardial ischemia-reperfusion injury.","method":"Cardiac-specific USP48 knockout and overexpression mouse models (STZ-induced diabetic), proteomics, RNA sequencing, CNN1 silencing rescue experiments, ERK1/2 pathway analysis, CXCL1/2 antibody blockade, infarct size measurement, mitochondrial damage assessment","journal":"Metabolism: Clinical and Experimental","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo genetic model with proteomics validation and mechanistic rescue; USP48–CNN1 stabilization inferred but direct deubiquitination of CNN1 not shown biochemically","pmids":["40513941"],"is_preprint":false}],"current_model":"CNN1 (calponin 1) is a smooth muscle-restricted actin-filament-associated protein that inhibits actin-activated myosin ATPase through direct actin binding; this inhibitory activity is abolished by PKC- or CaM-kinase II-mediated phosphorylation of CNN1, which reduces its actin affinity. CNN1 also functions as a scaffold/adapter linking MAPK and PKC-ε to the plasma membrane, associates with the cGMP kinase signaling complex, stabilizes the PDLIM7/ROCK1/MLC pathway in cancer-associated fibroblasts to modulate matrix stiffness, is regulated transcriptionally by a single intronic CArG/SRF element and post-transcriptionally by miRNAs (miR-106b-5p, miR-1274B, miR-330) and epigenetically by LINC00337-recruited DNMT1, and is stabilized post-translationally by the deubiquitinase USP48; collectively, CNN1 suppresses smooth muscle proliferation, neointimal formation, cancer cell invasion/metastasis, and cardiomyocyte injury signaling via ERK1/2-CXCL1/2."},"narrative":{"teleology":[{"year":1988,"claim":"Identification of calponin as a novel thin-filament-associated protein in smooth muscle established its candidacy as a contractile regulator analogous to troponin T.","evidence":"Biochemical purification from bovine aortic smooth muscle with binding assays for actin, tropomyosin, and calmodulin","pmids":["2455687"],"confidence":"High","gaps":["Functional consequence of actin binding on contractility not yet tested","Tissue distribution and isoform diversity unknown"]},{"year":1990,"claim":"Reconstitution experiments resolved CNN1's core molecular function: direct actin binding inhibits myosin ATPase, and PKC/CaM-kinase II phosphorylation serves as the off-switch by reducing actin affinity.","evidence":"Reconstituted actomyosin ATPase assay with in vitro phosphorylation and binding measurements","pmids":["2161834"],"confidence":"High","gaps":["Phosphorylation sites not mapped","In vivo relevance of ATPase inhibition not demonstrated","Structural basis of actin interaction unknown"]},{"year":1995,"claim":"Cloning the human CNN1 cDNA confirmed smooth muscle-exclusive expression and mapped the gene to chromosome 19p13, enabling genetic and regulatory studies.","evidence":"cDNA cloning from human aorta, RT-PCR tissue profiling, FISH chromosomal mapping","pmids":["8526917"],"confidence":"High","gaps":["Transcriptional regulatory elements not identified","No functional genomics in disease context"]},{"year":1997,"claim":"Discovery that CNN1 co-immunoprecipitates with MAPK and PKC-ε and co-redistributes to the plasma membrane upon agonist stimulation expanded CNN1's role beyond ATPase inhibition to a signaling scaffold in smooth muscle cells.","evidence":"Co-immunoprecipitation, overlay assay for direct MAPK binding, confocal microscopy of phenylephrine-stimulated VSMCs","pmids":["9312127"],"confidence":"Medium","gaps":["Binding domains on CNN1 for MAPK/PKC-ε not mapped","Functional consequence of scaffolding on downstream signaling not tested","Single lab finding without independent replication"]},{"year":2003,"claim":"Identification of CNN1 as a component of a cGMP kinase I signaling complex (with IP3RI, IRAG, phospholamban, α-actin) in tracheal smooth muscle placed CNN1 within a second signal transduction node beyond the MAPK/PKC-ε axis.","evidence":"MALDI-TOF mass spectrometry identification and reciprocal co-immunoprecipitation with multiple antibodies","pmids":["12480535"],"confidence":"Medium","gaps":["Functional role of CNN1 within the cGMP kinase complex not determined","Whether CNN1 is stoichiometric or substoichiometric in the complex is unknown"]},{"year":2011,"claim":"In vivo transgenic studies demonstrated that CNN1 overexpression suppresses neointimal formation after arterial injury and that a single intronic CArG/SRF element is necessary for smooth muscle-restricted CNN1 transcription, connecting gene regulation to vascular disease phenotype.","evidence":"BAC transgenic mice with CArG element mutations, femoral artery injury model, lacZ reporter","pmids":["21817093"],"confidence":"High","gaps":["Sufficiency of CArG element for full expression not established","Mechanism by which CNN1 suppresses neointimal formation not defined"]},{"year":2015,"claim":"Discovery that XBP1s induces miR-1274B to degrade CNN1 mRNA, promoting SMC proliferation and neointimal formation, established the first post-transcriptional regulatory axis controlling CNN1 levels in vascular disease.","evidence":"SMC knockdown/overexpression, miRNA target validation, femoral artery injury mouse model","pmids":["26315405"],"confidence":"Medium","gaps":["Direct miR-1274B binding site on CNN1 3'UTR not mapped by mutagenesis","TGF-β3 counterregulatory mechanism incompletely defined"]},{"year":2020,"claim":"Multiple studies revealed CNN1 as a convergent tumor suppressor target silenced by distinct miRNAs (miR-106b-5p in breast cancer) and by DNMT1-mediated promoter methylation (recruited by LINC00337 in colorectal cancer), with functional consequences for proliferation, invasion, and metastasis.","evidence":"Dual-luciferase 3'UTR assays, bisulfite/methylation-specific PCR, gain/loss-of-function in cancer cell lines, nude mouse xenograft and lung metastasis models","pmids":["31986487","33328585"],"confidence":"Medium","gaps":["Direct downstream effectors of CNN1 tumor suppression not identified in most cancer types","Whether CNN1 acts cell-autonomously or through microenvironment remodeling in tumors is unresolved"]},{"year":2022,"claim":"In cancer-associated fibroblasts, CNN1 stabilizes PDLIM7 by blocking NEDD4-1-mediated ubiquitination, activating ROCK1/MLC-dependent matrix stiffening that drives YAP activation and chemoresistance in adjacent tumor cells — defining a non-cell-autonomous tumor-promoting mechanism distinct from CNN1's cell-autonomous tumor suppressor role.","evidence":"Co-immunoprecipitation, ubiquitination assay, atomic force microscopy in 3D culture, mouse gastric cancer model","pmids":["36423735"],"confidence":"Medium","gaps":["Binding domain between CNN1 and PDLIM7 not mapped","Whether this CAF mechanism applies beyond gastric cancer is unknown","Reconciliation with CNN1's tumor-suppressive roles in epithelial cells not addressed"]},{"year":2024,"claim":"Expanding CNN1 biology beyond smooth muscle, UTX was shown to bind CNN1 to orchestrate trauma-induced endothelial cell senescence and SASP, while a MEG3/miR-330/CNN1 ceRNA axis was validated in breast cancer, and CNN1 knockdown impaired satellite cell proliferation and lipid accumulation in avian skeletal muscle.","evidence":"Spinal cord injury mouse model with endothelial UTX deletion; RIP and luciferase validation of MEG3–miR-330–CNN1 axis; siRNA knockdown in chicken satellite cells","pmids":["38528029","38240701","38669818"],"confidence":"Medium","gaps":["Nature of the UTX–CNN1 interaction (direct vs. transcriptional) not biochemically resolved","Satellite cell findings lack pathway placement and are in a non-mammalian model","Relative contribution of CNN1 to endothelial senescence versus smooth muscle phenotypes unclear"]},{"year":2025,"claim":"Identification of USP48 as a deubiquitinase that stabilizes CNN1 protein in cardiomyocytes, with the USP48–CNN1 axis suppressing ERK1/2-mediated CXCL1/2 release to protect against ischemia-reperfusion injury, extended CNN1's protective role to cardiac tissue.","evidence":"Cardiac-specific USP48 knockout/overexpression in diabetic mice, proteomics, CNN1 silencing rescue, ERK1/2 pathway and CXCL antibody blockade","pmids":["40513941"],"confidence":"Medium","gaps":["Direct deubiquitination of CNN1 by USP48 not demonstrated by in vitro reconstitution","Whether CNN1 is expressed endogenously in cardiomyocytes or only under stress is not established","Mechanism linking CNN1 to ERK1/2 inactivation not defined"]},{"year":null,"claim":"Key unresolved questions include the structural basis of CNN1's actin-binding and ATPase-inhibitory activity, the identity of CNN1 phosphorylation sites in vivo, how CNN1 toggles between tumor-suppressive (epithelial-autonomous) and tumor-promoting (CAF-mediated) roles, and whether its newly described cardiac and endothelial functions represent de novo expression or re-activation of a latent smooth muscle program.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of CNN1 bound to actin","In vivo phosphosite mapping not performed","Context-dependent tumor suppressor versus promoter mechanism unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,4]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,1,7]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,4]}],"pathway":[{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[0,1,7]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4,11,16]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[8,9,10,12,14]}],"complexes":["cGMP kinase I signaling complex"],"partners":["MAPK1","PRKCЕ","PDLIM7","USP48","KDM6A","PRKG1","ACTA2"],"other_free_text":[]},"mechanistic_narrative":"CNN1 (calponin-1) is a smooth muscle-restricted actin-filament-associated protein that inhibits actin-activated myosin MgATPase activity through direct actin binding, thereby fine-tuning smooth muscle contractility; this inhibition is abolished by PKC- or CaM-kinase II-mediated phosphorylation, which reduces actin affinity while preserving calmodulin and tropomyosin binding [PMID:2161834, PMID:26970176]. Beyond its contractile role, CNN1 functions as a scaffold linking MAPK and PKC-ε to the plasma membrane in vascular smooth muscle and participates in a cGMP kinase signaling complex in airway smooth muscle [PMID:9312127, PMID:12480535]. CNN1 expression is transcriptionally controlled by a single intronic CArG/SRF element and post-transcriptionally regulated by multiple miRNAs (miR-1274B, miR-106b-5p, miR-330) and LINC00337-recruited DNMT1 promoter methylation; CNN1 overexpression suppresses neointimal formation after vascular injury and inhibits cancer cell invasion and metastasis across multiple tumor types [PMID:21817093, PMID:26315405, PMID:31986487, PMID:33328585]. CNN1 protein is stabilized post-translationally by USP48, and the USP48–CNN1 axis protects cardiomyocytes from ischemia-reperfusion injury by inactivating ERK1/2-dependent CXCL1/2 release [PMID:40513941]."},"prefetch_data":{"uniprot":{"accession":"P51911","full_name":"Calponin-1","aliases":["Basic calponin","Calponin H1, smooth muscle"],"length_aa":297,"mass_kda":33.2,"function":"Thin filament-associated protein that is implicated in the regulation and modulation of smooth muscle contraction. It is capable of binding to actin, calmodulin and tropomyosin. The interaction of calponin with actin inhibits the actomyosin Mg-ATPase activity (By similarity)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/P51911/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNN1","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CNN1","total_profiled":1310},"omim":[{"mim_id":"611795","title":"MICRO RNA 145; MIR145","url":"https://www.omim.org/entry/611795"},{"mim_id":"610674","title":"SPERM FLAGELLAR PROTEIN 1; SPEF1","url":"https://www.omim.org/entry/610674"},{"mim_id":"610077","title":"REGULATOR OF CELL CYCLE; RGCC","url":"https://www.omim.org/entry/610077"},{"mim_id":"609922","title":"EH DOMAIN-BINDING PROTEIN 1; EHBP1","url":"https://www.omim.org/entry/609922"},{"mim_id":"608667","title":"NIPPED-B-LIKE; NIPBL","url":"https://www.omim.org/entry/608667"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Actin filaments","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"blood vessel","ntpm":2087.4},{"tissue":"intestine","ntpm":2743.0},{"tissue":"smooth muscle","ntpm":2059.2}],"url":"https://www.proteinatlas.org/search/CNN1"},"hgnc":{"alias_symbol":["SMCC","Sm-Calp"],"prev_symbol":[]},"alphafold":{"accession":"P51911","domains":[{"cath_id":"1.10.418.10","chopping":"12-133","consensus_level":"high","plddt":90.9106,"start":12,"end":133}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P51911","model_url":"https://alphafold.ebi.ac.uk/files/AF-P51911-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P51911-F1-predicted_aligned_error_v6.png","plddt_mean":71.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNN1","jax_strain_url":"https://www.jax.org/strain/search?query=CNN1"},"sequence":{"accession":"P51911","fasta_url":"https://rest.uniprot.org/uniprotkb/P51911.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P51911/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P51911"}},"corpus_meta":[{"pmid":"10198638","id":"PMC_10198638","title":"Identity between TRAP and SMCC 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stabilizes the actin cytoskeleton, thereby fine-tuning smooth muscle contractility. Phosphorylation regulates calponin function.\",\n      \"method\": \"Review synthesizing three decades of biochemical and cell biological studies including in vitro actin-binding assays, myosin ATPase inhibition assays, and phosphorylation studies\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assays replicated across multiple labs over decades\",\n      \"pmids\": [\"26970176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"miR-106b-5p directly binds the 3'UTR of CNN1 mRNA (validated by dual-luciferase assay) and suppresses CNN1 expression, thereby activating the Rho/ROCK1 signaling pathway to promote breast cancer migration, invasion, and lung metastasis.\",\n      \"method\": \"Dual-luciferase reporter assay, transcript half-life assay, in vivo lung metastasis assay, CNN1 overexpression/knockdown with cell functional readouts, Rho/ROCK1 inhibition assay\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods from single lab; direct 3'UTR binding validated\",\n      \"pmids\": [\"31986487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LINC00337 recruits DNMT1 to the CNN1 promoter region, causing hypermethylation and transcriptional silencing of CNN1 in colorectal cancer cells, which promotes tumor angiogenesis.\",\n      \"method\": \"Bisulfite sequencing PCR (BSP), methylation-specific PCR (MSP), loss- and gain-of-function experiments, in vitro tube formation assay, in vivo xenograft tumor model with MVD and VEGF quantification\",\n      \"journal\": \"Cancer gene therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic epigenetic recruitment demonstrated with multiple methods from single lab\",\n      \"pmids\": [\"33328585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CNN1 overexpression in lung squamous cell carcinoma cells activates TIMP2, which in turn upregulates DKK1 and suppresses the Wnt/β-catenin/c-myc signaling pathway, inhibiting EMT, invasion, and migration.\",\n      \"method\": \"Transient transfection overexpression, wound healing assay, Transwell invasion assay, RT-qPCR, Western blotting, immunofluorescence\",\n      \"journal\": \"Experimental and therapeutic medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, correlative pathway placement without direct protein-protein interaction evidence\",\n      \"pmids\": [\"34178128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CNN1 overexpression in bladder cancer cells decreases protein levels of glycolysis-related proteins GLUT1, PKM2, and LDHA, and downregulates HIF-1α and PDK1, thereby inhibiting metabolic reprogramming (Warburg effect), as well as cell proliferation, invasion, and migration.\",\n      \"method\": \"CNN1 overexpression, CCK8 proliferation assay, Transwell invasion/migration assay, glucose absorption/lactate secretion/ATP level kits, Western blotting, RT-PCR, nude mouse xenograft model, IHC for Ki-67 and CNN1\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, no direct interaction shown between CNN1 and HIF-1α\",\n      \"pmids\": [\"35903683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Lysine demethylase 6A (UTX/KDM6A) binds to CNN1 to form an epigenetic regulatory axis that orchestrates trauma-induced senescence and SASP secretion in spinal cord microvascular endothelial cells; UTX upregulation induces CNN1-dependent SCMEC senescence and amplifies proinflammatory SASP secretion, while UTX deletion prevents these effects and promotes neurological recovery.\",\n      \"method\": \"UTX knockout/overexpression in endothelial cells, co-immunoprecipitation/binding experiments, SASP factor measurement, in vivo spinal cord injury model, senescence assays, local senolytic drug delivery\",\n      \"journal\": \"Bone research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic KO with mechanistic binding data and defined cellular phenotype\",\n      \"pmids\": [\"38528029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP48 (ubiquitin-specific protease 48) stabilizes and upregulates CNN1 protein (preventing its degradation), and the USP48/CNN1 axis suppresses ERK1/2 pathway activity to reduce CXCL1 and CXCL2 release, thereby protecting against diabetes-aggravated myocardial ischemia/reperfusion injury.\",\n      \"method\": \"Proteomics, cardiac-specific USP48 knockout and overexpression mouse models, CNN1 silencing rescue experiments, RNA sequencing, Western blotting, in vitro H9c2 hypoxia/reoxygenation model, coronary artery occlusion/reperfusion model\",\n      \"journal\": \"Metabolism: clinical and experimental\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including proteomics, genetic KO/OE, and rescue experiments from single lab\",\n      \"pmids\": [\"40513941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"lncRNA MEG3 directly binds miR-330 (validated by dual-luciferase assay and RNA immunoprecipitation), acting as a competing endogenous RNA to derepress CNN1 expression; this MEG3/miR-330/CNN1 axis suppresses breast cancer cell viability, migration, invasion, and promotes apoptosis.\",\n      \"method\": \"Dual-luciferase reporter assay, RNA immunoprecipitation (RIP), overexpression/knockdown in MCF-7 and BT-474 cells, rescue experiments, flow cytometry\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct RNA binding validated by two orthogonal methods with functional rescue\",\n      \"pmids\": [\"38240701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UTMD-mediated CNN1 overexpression induces ferroptosis in colorectal cancer cells by activating p53 signaling, which directly binds the SLC7A11 promoter to suppress SLC7A11 transcription; rescue experiments confirmed that CNN1 overexpression acts through the p53-SLC7A11 pathway.\",\n      \"method\": \"UTMD-assisted gene transfection, p53 promoter-binding assay, SLC7A11 expression analysis, rescue experiments with p53-SLC7A11 pathway, proliferation and apoptosis assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, indirect pathway placement; direct CNN1-p53 interaction not demonstrated\",\n      \"pmids\": [\"40602187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CNN1 knockdown via siRNA in chicken satellite cells reduces proliferation and differentiation and decreases lipid accumulation during differentiation, establishing a role for CNN1 in skeletal muscle satellite cell function.\",\n      \"method\": \"siRNA knockdown in primary satellite cells, proliferation assay, differentiation assay, lipid accumulation measurement\",\n      \"journal\": \"Poultry science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single KD approach in avian satellite cells; no molecular mechanism elucidated\",\n      \"pmids\": [\"38669818\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNN1 (calponin 1) is a smooth muscle-specific actin filament-associated protein that inhibits actin-activated myosin ATPase and stabilizes the actin cytoskeleton to fine-tune contractility; its expression is epigenetically silenced in multiple cancers via DNMT1 recruitment and miRNA-mediated suppression (miR-106b-5p, miR-330), and when present, CNN1 suppresses tumor cell migration/invasion through inhibition of Rho/ROCK1 and Wnt/β-catenin signaling, reduces glycolytic reprogramming via HIF-1α downregulation, and in cardiomyocytes is stabilized by the deubiquitinase USP48 to suppress ERK1/2-mediated CXCL1/2 release, while an epigenetic UTX/KDM6A–CNN1 axis governs endothelial cell senescence and SASP secretion after injury.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n\n**KEEP (canonical CNN1 / calponin 1 protein):**\n- [5] Liu R, Jin JP 2016 — calponin isoforms review (KEEP)\n- [9] Wang Z et al. 2020 — miR-106b-5p/CNN1/Rho-ROCK1 (KEEP)\n- [11] Xu X et al. 2020 — LINC00337/DNMT1/CNN1 (KEEP)\n- [14] Liu W et al. 2021 — CNN1/TIMP2/DKK1/Wnt (KEEP)\n- [19] Zhang Z et al. 2022 — CNN1/HIF-1α in bladder cancer (KEEP)\n- [20] Li C et al. 2024 — Kdm6a-CNN1 axis (KEEP)\n- [28] Yi D et al. 2024 — MEG3/miR-330/CNN1 (KEEP)\n- [30] Velleman SG et al. 2024 — CNN1 in chicken satellite cells (KEEP — ortholog)\n- [32] Su JB et al. 2025 — USP48/CNN1 cardioprotection (KEEP)\n- [33] Wang L et al. 2025 — CNN1/p53/SLC7A11/ferroptosis (KEEP) — NOTE: abstract conflates \"CNN1\" with \"CyclinD1\" — likely alias collision; EXCLUDE\n- [gene2pubmed-10] Winder SJ, Walsh MP 1990 — calponin inhibits actomyosin ATPase (KEEP)\n- [gene2pubmed-13] Takahashi K et al. 1988 — calponin discovery (KEEP)\n- [gene2pubmed-15] Menice CB et al. 1997 — calponin/MAPK signaling (KEEP)\n- [gene2pubmed-19] Lu Y et al. 2022 — CNN1/PDLIM7/NEDD4-1/ROCK1 in CAFs (KEEP)\n- [gene2pubmed-21] Zeng L et al. 2015 — XBP1s/miR-1274B/CNN1 in SMC (KEEP)\n- [gene2pubmed-22] Maguchi M et al. 1995 — cloning of human calponin (KEEP)\n- [gene2pubmed-23] Koller A et al. 2003 — calponin H1 in cGMP kinase complex (KEEP)\n- [gene2pubmed-24] Long X et al. 2011 — CNN1 CArG elements, neointima (KEEP)\n- [gene2pubmed-4] Shah NM et al. 1996 — TGFβ/BMP2 → smooth muscle differentiation with calponin as marker (borderline; calponin used as marker, not mechanistic focus — EXCLUDE)\n- [gene2pubmed-18] Russell TD et al. 2015 — myoepithelial marker IHC (EXCLUDE — IHC marker study)\n- [gene2pubmed-20] Yamamura H et al. 1998 — osteosarcoma expression/prognosis (EXCLUDE — prognostic)\n- [gene2pubmed-25] Fukui Y et al. 1997 — h2-calponin in keratinocytes (EXCLUDE — about CNN2 isoform)\n- [gene2pubmed-27] Sasaki Y et al. 2002 — IHC/prognosis in HCC (EXCLUDE)\n- [gene2pubmed-30] Marsh WL et al. 2009 — IHC marker (EXCLUDE)\n\n**EXCLUDE (alias collisions / off-target):**\n- [1–4,6,8] TRAP/SMCC complex papers — SMCC here = SRB/MED-containing cofactor complex, not CNN1\n- [7] Bock LJ et al. 2012 — yeast Cnn1 = CENP-T ortholog at kinetochore — different protein; context (kinetochore, chromosome segregation) is fundamentally incompatible with mammalian CNN1 (calponin 1, actin-binding smooth muscle protein). SYMBOL COLLISION — EXCLUDE\n- [15] Hinshaw SM, Harrison SC 2020 — yeast Cnn1/CENP-T kinetochore — SYMBOL COLLISION — EXCLUDE\n- [18] Thapa KS et al. 2015 — yeast Cnn1(CENP-T) kinetochore — SYMBOL COLLISION — EXCLUDE\n- [10] Hangeland et al. 1997 — SMCC linker chemistry — EXCLUDE\n- [12] Li F et al. 2015 — PEI-SMCC-TAT gene delivery — EXCLUDE\n- [13] Wang X et al. 2020 — GAS5/PTEN in SMCC-7721 cells (hepatoma cell line, not CNN1 gene) — EXCLUDE\n- [16] AFM on SMCC-7721 cells — EXCLUDE\n- [17] SMCC biochip — EXCLUDE\n- [21] Collagen/Sulfo-SMCC scaffolds — EXCLUDE\n- [22] 1,4-Naphthoquinone labeled CNN1 — this is a chemical compound named CNN1, not the gene — SYMBOL COLLISION — EXCLUDE\n- [23] Streptomyces cnn1 mutant — microbial gene — SYMBOL COLLISION — EXCLUDE\n- [24–27,29,34,35] SMCC-7721 hepatoma cell line studies — EXCLUDE\n- [31] SMCC clustering algorithm — EXCLUDE\n- [36] NucA-SMCC-DM1 conjugates — EXCLUDE\n- [37–43] Preprints: most involve SMCC as statistical measure or unrelated; [38] mentions CNN1 as VSMC differentiation marker only; [39] mentions Cnn1 as SMC marker only — these don't provide mechanistic findings about CNN1 beyond marker expression — EXCLUDE for mechanism purposes\n- [33] Wang L et al. 2025 — abstract states \"CyclinD1 (CNN1)\" — this is a symbol collision (CCND1/CyclinD1 ≠ CNN1/calponin1) — EXCLUDE\n- [gene2pubmed-1,2,3,5,6,7,8,9,11,12,14,16,17,26,28,29] — genomics/proteomics/GWAS resources, not CNN1-specific mechanistic — EXCLUDE\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1988,\n      \"finding\": \"Calponin was purified from bovine aortic smooth muscle as a novel troponin T-like protein that binds actin, tropomyosin, and calmodulin. It was identified as a component of the smooth muscle thin filament with potential regulatory roles in the contractile apparatus.\",\n      \"method\": \"Protein purification, SDS-PAGE, isoelectric focusing, calmodulin-binding assay, tropomyosin-binding assay, immunological cross-reactivity with troponin T\",\n      \"journal\": \"Hypertension\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original biochemical purification and characterization with multiple binding assays; foundational discovery paper\",\n      \"pmids\": [\"2455687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Calponin (CNN1) inhibits actin-activated myosin MgATPase activity in a reconstituted smooth muscle contractile system. Inhibition requires calponin–actin interaction (not calponin–tropomyosin interaction) and is independent of Ca2+ or myosin phosphorylation state. Phosphorylation of calponin by protein kinase C or Ca2+/calmodulin-dependent protein kinase II abolishes ATPase inhibition and greatly reduces actin binding, while calmodulin- and tropomyosin-binding capabilities are retained.\",\n      \"method\": \"Reconstituted actomyosin ATPase assay, in vitro phosphorylation by PKC and CaM-kinase II, actin-binding assay, calmodulin-binding assay, tropomyosin-binding assay\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mechanistic dissection of actin-binding requirement; foundational mechanism paper replicated across field\",\n      \"pmids\": [\"2161834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Human basic calponin (CNN1) cDNA was cloned from human aorta, encoding 297 amino acids (33,169 Da). CNN1 mRNA is expressed exclusively in smooth muscle tissues. The CNN1 gene localizes to chromosome 19p13.1–13.2 by fluorescent in situ hybridization.\",\n      \"method\": \"cDNA cloning, RT-PCR tissue expression profiling, fluorescent in situ hybridization (FISH)\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct molecular cloning and chromosomal mapping\",\n      \"pmids\": [\"8526917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"In differentiated vascular smooth muscle cells, calponin co-immunoprecipitates with mitogen-activated protein kinase (MAPK) and with protein kinase C-epsilon (PKC-ε). An overlay assay confirmed direct interaction between MAPK and calponin, though MAPK does not phosphorylate calponin in vitro. During phenylephrine stimulation, calponin redistributes to the cell membrane coincident with MAPK and PKC-ε membrane targeting, suggesting calponin acts as an adapter/scaffold linking these kinases to the plasma membrane.\",\n      \"method\": \"Co-immunoprecipitation, overlay assay, in vitro kinase assay, high-resolution digital confocal microscopy\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP and overlay with functional imaging; single lab study\",\n      \"pmids\": [\"9312127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Calponin H1 (CNN1) is a component of a cGMP kinase signaling complex in tracheal smooth muscle membranes, co-immunoprecipitating with IP3 receptor I, IRAG, cGMP kinase I, alpha-actin, and phospholamban. The complex was validated by mutual co-immunoprecipitation using antibodies to each member protein.\",\n      \"method\": \"Protein complex purification, MALDI-TOF mass spectrometry, MS/MS sequencing, immunoprecipitation with multiple antibodies, Western blotting\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP across multiple antibodies identifying complex membership; single lab\",\n      \"pmids\": [\"12480535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CNN1 overexpression suppresses neointimal formation following arterial injury in transgenic mice. A single intronic CArG element (serum response factor binding site) is necessary but insufficient for correct smooth muscle cell-restricted expression of CNN1 in vivo; deletion of orthologous intronic sequences abolishes endogenous Cnn1 promoter activity.\",\n      \"method\": \"Transgenic mouse model (BAC transgenics), BAC recombineering with CArG element mutations, femoral artery injury model, immunohistochemistry, Western blotting, lacZ reporter assay\",\n      \"journal\": \"Arteriosclerosis, Thrombosis, and Vascular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo gain-of-function with defined phenotypic readout, CArG mutation series confirms regulatory element requirement\",\n      \"pmids\": [\"21817093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"XBP1s (spliced XBP1) decreases CNN1 expression in smooth muscle cells by directing transcription of miR-1274B, which targets CNN1 mRNA for degradation, thereby promoting SMC proliferation. TGF-β3 (but not TGF-β1 or TGF-β2) attenuates XBP1s-induced CNN1 decrease and SMC proliferation. XBP1 deficiency in SMCs abrogates neointimal formation in vivo.\",\n      \"method\": \"SMC knockdown/overexpression, miRNA target validation, femoral artery injury mouse model, proteomic analysis of culture media, RT-PCR, Western blotting\",\n      \"journal\": \"Arteriosclerosis, Thrombosis, and Vascular Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo model with mechanistic dissection of miRNA-mediated CNN1 regulation; single lab\",\n      \"pmids\": [\"26315405\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CNN1 (calponin 1) is specifically expressed in smooth muscle cells and functions to inhibit actin-activated myosin ATPase and stabilize the actin cytoskeleton, fine-tuning smooth muscle contractility. Phosphorylation regulates CNN1 function. The CNN1 gene is one of three vertebrate calponin isoform genes, with smooth muscle-restricted expression.\",\n      \"method\": \"Review synthesizing in vitro ATPase assays, phosphorylation studies, expression profiling, and cytoskeletal analyses from multiple studies\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — review consolidating decades of replicated biochemical and cell biological data\",\n      \"pmids\": [\"26970176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"miR-106b-5p directly binds to the CNN1 mRNA 3'UTR (validated by dual-luciferase assay) and suppresses CNN1 expression, activating the Rho/ROCK1 signaling pathway to promote breast cancer cell survival, migration, invasion, adhesion, and in vivo lung metastasis.\",\n      \"method\": \"Dual-luciferase reporter assay, CNN1 overexpression/knockdown in breast cancer cell lines, cell viability/migration/invasion/adhesion assays, in vivo lung metastasis assay, transcript half-life assay, Rho/ROCK1 inhibition assay\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct 3'UTR binding validated, pathway inhibition rescue; single lab\",\n      \"pmids\": [\"31986487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LINC00337 recruits DNMT1 to the promoter region of CNN1, causing hypermethylation and transcriptional silencing of CNN1 in colorectal cancer. Reduced CNN1 expression promotes CRC cell proliferation, migration/invasion, and tumor angiogenesis (VEGF upregulation), whereas CNN1 overexpression suppresses these phenotypes in vitro and in vivo.\",\n      \"method\": \"Bisulfite sequencing PCR (BSP), methylation-specific PCR (MSP), loss/gain-of-function experiments, MTT assay, Transwell assay, tube formation assay, nude mouse xenograft, DNMT1 recruitment ChIP-like assay\",\n      \"journal\": \"Cancer Gene Therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — epigenetic mechanism validated by BSP/MSP with functional rescue; single lab\",\n      \"pmids\": [\"33328585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CNN1 overexpression in lung squamous cell carcinoma cells activates TIMP2 expression and inhibits the DKK1/Wnt/β-catenin/c-myc signaling pathway, suppressing invasion, migration, and epithelial-to-mesenchymal transition (EMT). Inhibition of DKK1 reverses these CNN1-mediated effects, restoring β-catenin and c-myc expression.\",\n      \"method\": \"CNN1 transient overexpression, GEPIA database correlation analysis, wound healing assay, Transwell invasion assay, RT-qPCR, Western blotting, immunofluorescence staining\",\n      \"journal\": \"Experimental and Therapeutic Medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — overexpression with pathway marker readout but no direct biochemical interaction; single lab\",\n      \"pmids\": [\"34178128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CNN1 in cancer-associated fibroblasts (CAFs) interacts with PDZ and LIM Domain 7 (PDLIM7) and prevents its degradation by the E3 ubiquitin ligase NEDD4-1, stabilizing PDLIM7 and thereby activating the ROCK1/MLC pathway. This increases matrix stiffness (CAF contraction), which in turn activates YAP in adjacent gastric cancer cells to confer 5-fluorouracil resistance.\",\n      \"method\": \"Co-immunoprecipitation, CAF knockdown in 3D culture and mouse GC model, atomic force microscopy for matrix stiffness, NEDD4-1 ubiquitination assay, YAP activity readout\",\n      \"journal\": \"Matrix Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP identifies binding partner, ubiquitination assay defines mechanism, in vivo rescue; single lab\",\n      \"pmids\": [\"36423735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CNN1 overexpression in bladder cancer cells suppresses glycolysis by decreasing protein levels of GLUT1, PKM2, and LDHA, with concomitant reduction of HIF-1α and PDK1 mRNA and protein levels, inhibiting tumor cell proliferation, invasion, and migration in vitro and in vivo.\",\n      \"method\": \"CNN1 overexpression in BC cell lines, CCK8 proliferation assay, Transwell assay, Western blotting, RT-PCR, glucose uptake/lactate secretion/ATP level kits, nude mouse xenograft, immunohistochemistry\",\n      \"journal\": \"Frontiers in Oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — overexpression with pathway marker readout; no direct biochemical interaction demonstrated; single lab\",\n      \"pmids\": [\"35903683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UTX (Kdm6a/lysine demethylase 6A) forms an epigenetic regulatory axis by binding to CNN1, orchestrating trauma-induced senescence of spinal cord microvascular endothelial cells (SCMECs) and their senescence-associated secretory phenotype (SASP). UTX upregulation after spinal cord injury induces CNN1-dependent SCMEC senescence and proinflammatory SASP release; UTX deletion protects SCMECs against senescence and promotes neurological recovery.\",\n      \"method\": \"Spinal cord injury mouse model, endothelial cell-specific UTX deletion, UTX overexpression, binding assay (UTX–CNN1 interaction), senescence markers, SASP cytokine profiling, functional neurological recovery assessment, senolytic drug delivery\",\n      \"journal\": \"Bone Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model with defined molecular interaction and functional phenotype; single lab\",\n      \"pmids\": [\"38528029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"lncRNA MEG3 acts as a ceRNA (competing endogenous RNA) by directly binding miR-330, preventing miR-330 from suppressing CNN1 expression in breast cancer. Overexpression of MEG3 inhibits breast cancer cell viability, migration, invasion, and promotes apoptosis; these effects are reversed by miR-330 mimic, which decreases CNN1 levels. Validated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP).\",\n      \"method\": \"Dual-luciferase reporter assay, RNA immunoprecipitation (RIP), CNN1/MEG3 overexpression/knockdown, cell viability, migration/invasion assays, flow cytometry for apoptosis and cell cycle\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct RNA–RNA interaction validated by RIP and luciferase; functional rescue experiments; single lab\",\n      \"pmids\": [\"38240701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CNN1 knockdown by siRNA in chicken (Ross 708 and Randombred) breast muscle satellite cells reduces proliferation, affects differentiation, and decreases lipid accumulation during differentiation, demonstrating a functional role for CNN1 in satellite cell-mediated muscle growth and regeneration.\",\n      \"method\": \"siRNA knockdown of CNN1, satellite cell isolation, proliferation assay, differentiation assay, lipid accumulation assay\",\n      \"journal\": \"Poultry Science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — clean knockdown with defined phenotype but no pathway placement; single lab, avian model\",\n      \"pmids\": [\"38669818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP48 (ubiquitin-specific protease 48) stabilizes and upregulates CNN1 protein in cardiomyocytes; CNN1 silencing abrogates the cardioprotective effects of USP48. The USP48/CNN1 axis inhibits release of CXCL1 and CXCL2 by inactivating the ERK1/2 pathway, protecting against diabetes-aggravated myocardial ischemia-reperfusion injury.\",\n      \"method\": \"Cardiac-specific USP48 knockout and overexpression mouse models (STZ-induced diabetic), proteomics, RNA sequencing, CNN1 silencing rescue experiments, ERK1/2 pathway analysis, CXCL1/2 antibody blockade, infarct size measurement, mitochondrial damage assessment\",\n      \"journal\": \"Metabolism: Clinical and Experimental\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model with proteomics validation and mechanistic rescue; USP48–CNN1 stabilization inferred but direct deubiquitination of CNN1 not shown biochemically\",\n      \"pmids\": [\"40513941\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNN1 (calponin 1) is a smooth muscle-restricted actin-filament-associated protein that inhibits actin-activated myosin ATPase through direct actin binding; this inhibitory activity is abolished by PKC- or CaM-kinase II-mediated phosphorylation of CNN1, which reduces its actin affinity. CNN1 also functions as a scaffold/adapter linking MAPK and PKC-ε to the plasma membrane, associates with the cGMP kinase signaling complex, stabilizes the PDLIM7/ROCK1/MLC pathway in cancer-associated fibroblasts to modulate matrix stiffness, is regulated transcriptionally by a single intronic CArG/SRF element and post-transcriptionally by miRNAs (miR-106b-5p, miR-1274B, miR-330) and epigenetically by LINC00337-recruited DNMT1, and is stabilized post-translationally by the deubiquitinase USP48; collectively, CNN1 suppresses smooth muscle proliferation, neointimal formation, cancer cell invasion/metastasis, and cardiomyocyte injury signaling via ERK1/2-CXCL1/2.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CNN1 (calponin 1) is a smooth muscle-enriched actin filament-associated protein that inhibits actin-activated myosin ATPase activity, stabilizes the actin cytoskeleton, and fine-tunes smooth muscle contractility, with its function regulated by phosphorylation [PMID:26970176]. CNN1 expression is frequently silenced in cancer through epigenetic mechanisms—including DNMT1-mediated promoter hypermethylation and miRNA-dependent mRNA degradation (miR-106b-5p, miR-330)—and its re-expression suppresses tumor cell migration and invasion by restraining Rho/ROCK1 signaling and Wnt/β-catenin pathways [PMID:31986487, PMID:33328585, PMID:38240701]. In cardiomyocytes, CNN1 protein is stabilized by the deubiquitinase USP48, and the USP48–CNN1 axis suppresses ERK1/2-mediated CXCL1/2 release to protect against myocardial ischemia/reperfusion injury [PMID:40513941]. In spinal cord microvascular endothelial cells, CNN1 functions downstream of the lysine demethylase UTX/KDM6A to orchestrate trauma-induced cellular senescence and senescence-associated secretory phenotype (SASP) secretion [PMID:38528029].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Decades of biochemical work established that CNN1 is a smooth muscle-specific actin-binding protein whose core activity—inhibition of actin-activated myosin ATPase—is regulated by phosphorylation, defining its fundamental role as a contractility modulator.\",\n      \"evidence\": \"Comprehensive review of in vitro actin-binding and myosin ATPase inhibition assays across multiple laboratories\",\n      \"pmids\": [\"26970176\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of in vivo kinases and phosphatases that dynamically regulate CNN1 activity remains incompletely mapped\",\n        \"Structural basis of CNN1-actin filament interaction at atomic resolution not determined\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"CNN1 was shown to be a direct target of post-transcriptional silencing in cancer: miR-106b-5p binds the CNN1 3′UTR to repress its expression in breast cancer, while LINC00337 recruits DNMT1 to hypermethylate the CNN1 promoter in colorectal cancer, revealing convergent epigenetic and miRNA-mediated mechanisms of CNN1 downregulation.\",\n      \"evidence\": \"Dual-luciferase 3′UTR reporter assay and in vivo metastasis model (breast cancer); bisulfite sequencing, methylation-specific PCR, and xenograft angiogenesis assay (colorectal cancer)\",\n      \"pmids\": [\"31986487\", \"33328585\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether DNMT1-mediated silencing and miRNA-mediated suppression cooperate in the same tumor type is untested\",\n        \"No chromatin immunoprecipitation data confirming DNMT1 occupancy at the CNN1 promoter independent of LINC00337\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Functional studies indicated that CNN1 re-expression in lung squamous cell carcinoma and bladder cancer suppresses invasion, EMT, and glycolytic reprogramming, linking CNN1 loss to activation of Wnt/β-catenin and HIF-1α pathways, though direct molecular interactions were not demonstrated.\",\n      \"evidence\": \"CNN1 overexpression with Transwell invasion, Western blot for Wnt/β-catenin components (lung cancer); CNN1 overexpression with glucose/lactate metabolic assays and xenograft (bladder cancer)\",\n      \"pmids\": [\"34178128\", \"35903683\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No direct physical interaction between CNN1 and Wnt or HIF-1α pathway components demonstrated\",\n        \"Both studies from single laboratories without independent replication\",\n        \"Mechanism by which a cytoskeletal protein influences nuclear transcription factors is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A UTX/KDM6A–CNN1 epigenetic axis was identified in spinal cord microvascular endothelial cells, where UTX upregulation drives CNN1-dependent cellular senescence and SASP secretion after injury, extending CNN1 function beyond contractile regulation to stress-induced senescence.\",\n      \"evidence\": \"UTX knockout/overexpression in endothelial cells, co-immunoprecipitation, SASP factor quantification, and in vivo spinal cord injury model\",\n      \"pmids\": [\"38528029\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether UTX directly demethylates the CNN1 locus or acts through an intermediary is not resolved\",\n        \"Generalizability of the UTX–CNN1 senescence axis to other endothelial beds or injury types untested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"lncRNA MEG3 was shown to competitively sponge miR-330, derepressing CNN1 and suppressing breast cancer cell viability and invasion, establishing a second validated ceRNA regulatory axis for CNN1.\",\n      \"evidence\": \"Dual-luciferase reporter and RNA immunoprecipitation in MCF-7 and BT-474 cells with functional rescue\",\n      \"pmids\": [\"38240701\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"In vivo validation of the MEG3/miR-330/CNN1 axis in tumor models not performed\",\n        \"Relative contribution of miR-330 versus miR-106b-5p to CNN1 suppression in breast cancer undefined\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"USP48 was identified as a deubiquitinase that stabilizes CNN1 protein in cardiomyocytes; the USP48–CNN1 axis suppresses ERK1/2-mediated CXCL1/2 chemokine release and protects against diabetes-aggravated myocardial ischemia/reperfusion injury, revealing post-translational control of CNN1 levels.\",\n      \"evidence\": \"Proteomics, cardiac-specific USP48 KO/OE mice, CNN1 silencing rescue, and coronary artery occlusion/reperfusion model\",\n      \"pmids\": [\"40513941\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The E3 ubiquitin ligase that targets CNN1 for degradation is not identified\",\n        \"Whether USP48-mediated CNN1 stabilization occurs in smooth muscle or endothelial cells is unknown\",\n        \"Direct deubiquitination of CNN1 by USP48 (in vitro reconstitution) not shown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of CNN1–actin interaction, identity of the E3 ligase targeting CNN1, and the molecular mechanism by which a cytoskeletal regulatory protein influences nuclear signaling pathways (Wnt, HIF-1α, p53-SLC7A11) in cancer contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of CNN1 bound to actin filaments\",\n        \"Direct interaction between CNN1 and nuclear transcription factors/pathways not demonstrated\",\n        \"Physiological relevance of CNN1 in non-smooth-muscle tissues beyond overexpression models is poorly defined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 5, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"USP48\",\n      \"UTX\",\n      \"DNMT1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CNN1 (calponin-1) is a smooth muscle-restricted actin-filament-associated protein that inhibits actin-activated myosin MgATPase activity through direct actin binding, thereby fine-tuning smooth muscle contractility; this inhibition is abolished by PKC- or CaM-kinase II-mediated phosphorylation, which reduces actin affinity while preserving calmodulin and tropomyosin binding [PMID:2161834, PMID:26970176]. Beyond its contractile role, CNN1 functions as a scaffold linking MAPK and PKC-ε to the plasma membrane in vascular smooth muscle and participates in a cGMP kinase signaling complex in airway smooth muscle [PMID:9312127, PMID:12480535]. CNN1 expression is transcriptionally controlled by a single intronic CArG/SRF element and post-transcriptionally regulated by multiple miRNAs (miR-1274B, miR-106b-5p, miR-330) and LINC00337-recruited DNMT1 promoter methylation; CNN1 overexpression suppresses neointimal formation after vascular injury and inhibits cancer cell invasion and metastasis across multiple tumor types [PMID:21817093, PMID:26315405, PMID:31986487, PMID:33328585]. CNN1 protein is stabilized post-translationally by USP48, and the USP48–CNN1 axis protects cardiomyocytes from ischemia-reperfusion injury by inactivating ERK1/2-dependent CXCL1/2 release [PMID:40513941].\",\n  \"teleology\": [\n    {\n      \"year\": 1988,\n      \"claim\": \"Identification of calponin as a novel thin-filament-associated protein in smooth muscle established its candidacy as a contractile regulator analogous to troponin T.\",\n      \"evidence\": \"Biochemical purification from bovine aortic smooth muscle with binding assays for actin, tropomyosin, and calmodulin\",\n      \"pmids\": [\"2455687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Functional consequence of actin binding on contractility not yet tested\",\n        \"Tissue distribution and isoform diversity unknown\"\n      ]\n    },\n    {\n      \"year\": 1990,\n      \"claim\": \"Reconstitution experiments resolved CNN1's core molecular function: direct actin binding inhibits myosin ATPase, and PKC/CaM-kinase II phosphorylation serves as the off-switch by reducing actin affinity.\",\n      \"evidence\": \"Reconstituted actomyosin ATPase assay with in vitro phosphorylation and binding measurements\",\n      \"pmids\": [\"2161834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Phosphorylation sites not mapped\",\n        \"In vivo relevance of ATPase inhibition not demonstrated\",\n        \"Structural basis of actin interaction unknown\"\n      ]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Cloning the human CNN1 cDNA confirmed smooth muscle-exclusive expression and mapped the gene to chromosome 19p13, enabling genetic and regulatory studies.\",\n      \"evidence\": \"cDNA cloning from human aorta, RT-PCR tissue profiling, FISH chromosomal mapping\",\n      \"pmids\": [\"8526917\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Transcriptional regulatory elements not identified\",\n        \"No functional genomics in disease context\"\n      ]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Discovery that CNN1 co-immunoprecipitates with MAPK and PKC-ε and co-redistributes to the plasma membrane upon agonist stimulation expanded CNN1's role beyond ATPase inhibition to a signaling scaffold in smooth muscle cells.\",\n      \"evidence\": \"Co-immunoprecipitation, overlay assay for direct MAPK binding, confocal microscopy of phenylephrine-stimulated VSMCs\",\n      \"pmids\": [\"9312127\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Binding domains on CNN1 for MAPK/PKC-ε not mapped\",\n        \"Functional consequence of scaffolding on downstream signaling not tested\",\n        \"Single lab finding without independent replication\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identification of CNN1 as a component of a cGMP kinase I signaling complex (with IP3RI, IRAG, phospholamban, α-actin) in tracheal smooth muscle placed CNN1 within a second signal transduction node beyond the MAPK/PKC-ε axis.\",\n      \"evidence\": \"MALDI-TOF mass spectrometry identification and reciprocal co-immunoprecipitation with multiple antibodies\",\n      \"pmids\": [\"12480535\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional role of CNN1 within the cGMP kinase complex not determined\",\n        \"Whether CNN1 is stoichiometric or substoichiometric in the complex is unknown\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"In vivo transgenic studies demonstrated that CNN1 overexpression suppresses neointimal formation after arterial injury and that a single intronic CArG/SRF element is necessary for smooth muscle-restricted CNN1 transcription, connecting gene regulation to vascular disease phenotype.\",\n      \"evidence\": \"BAC transgenic mice with CArG element mutations, femoral artery injury model, lacZ reporter\",\n      \"pmids\": [\"21817093\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Sufficiency of CArG element for full expression not established\",\n        \"Mechanism by which CNN1 suppresses neointimal formation not defined\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Discovery that XBP1s induces miR-1274B to degrade CNN1 mRNA, promoting SMC proliferation and neointimal formation, established the first post-transcriptional regulatory axis controlling CNN1 levels in vascular disease.\",\n      \"evidence\": \"SMC knockdown/overexpression, miRNA target validation, femoral artery injury mouse model\",\n      \"pmids\": [\"26315405\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct miR-1274B binding site on CNN1 3'UTR not mapped by mutagenesis\",\n        \"TGF-β3 counterregulatory mechanism incompletely defined\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Multiple studies revealed CNN1 as a convergent tumor suppressor target silenced by distinct miRNAs (miR-106b-5p in breast cancer) and by DNMT1-mediated promoter methylation (recruited by LINC00337 in colorectal cancer), with functional consequences for proliferation, invasion, and metastasis.\",\n      \"evidence\": \"Dual-luciferase 3'UTR assays, bisulfite/methylation-specific PCR, gain/loss-of-function in cancer cell lines, nude mouse xenograft and lung metastasis models\",\n      \"pmids\": [\"31986487\", \"33328585\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct downstream effectors of CNN1 tumor suppression not identified in most cancer types\",\n        \"Whether CNN1 acts cell-autonomously or through microenvironment remodeling in tumors is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"In cancer-associated fibroblasts, CNN1 stabilizes PDLIM7 by blocking NEDD4-1-mediated ubiquitination, activating ROCK1/MLC-dependent matrix stiffening that drives YAP activation and chemoresistance in adjacent tumor cells — defining a non-cell-autonomous tumor-promoting mechanism distinct from CNN1's cell-autonomous tumor suppressor role.\",\n      \"evidence\": \"Co-immunoprecipitation, ubiquitination assay, atomic force microscopy in 3D culture, mouse gastric cancer model\",\n      \"pmids\": [\"36423735\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Binding domain between CNN1 and PDLIM7 not mapped\",\n        \"Whether this CAF mechanism applies beyond gastric cancer is unknown\",\n        \"Reconciliation with CNN1's tumor-suppressive roles in epithelial cells not addressed\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Expanding CNN1 biology beyond smooth muscle, UTX was shown to bind CNN1 to orchestrate trauma-induced endothelial cell senescence and SASP, while a MEG3/miR-330/CNN1 ceRNA axis was validated in breast cancer, and CNN1 knockdown impaired satellite cell proliferation and lipid accumulation in avian skeletal muscle.\",\n      \"evidence\": \"Spinal cord injury mouse model with endothelial UTX deletion; RIP and luciferase validation of MEG3–miR-330–CNN1 axis; siRNA knockdown in chicken satellite cells\",\n      \"pmids\": [\"38528029\", \"38240701\", \"38669818\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Nature of the UTX–CNN1 interaction (direct vs. transcriptional) not biochemically resolved\",\n        \"Satellite cell findings lack pathway placement and are in a non-mammalian model\",\n        \"Relative contribution of CNN1 to endothelial senescence versus smooth muscle phenotypes unclear\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of USP48 as a deubiquitinase that stabilizes CNN1 protein in cardiomyocytes, with the USP48–CNN1 axis suppressing ERK1/2-mediated CXCL1/2 release to protect against ischemia-reperfusion injury, extended CNN1's protective role to cardiac tissue.\",\n      \"evidence\": \"Cardiac-specific USP48 knockout/overexpression in diabetic mice, proteomics, CNN1 silencing rescue, ERK1/2 pathway and CXCL antibody blockade\",\n      \"pmids\": [\"40513941\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct deubiquitination of CNN1 by USP48 not demonstrated by in vitro reconstitution\",\n        \"Whether CNN1 is expressed endogenously in cardiomyocytes or only under stress is not established\",\n        \"Mechanism linking CNN1 to ERK1/2 inactivation not defined\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of CNN1's actin-binding and ATPase-inhibitory activity, the identity of CNN1 phosphorylation sites in vivo, how CNN1 toggles between tumor-suppressive (epithelial-autonomous) and tumor-promoting (CAF-mediated) roles, and whether its newly described cardiac and endothelial functions represent de novo expression or re-activation of a latent smooth muscle program.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of CNN1 bound to actin\",\n        \"In vivo phosphosite mapping not performed\",\n        \"Context-dependent tumor suppressor versus promoter mechanism unexplained\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 11, 16]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [8, 9, 10, 12, 14]}\n    ],\n    \"complexes\": [\n      \"cGMP kinase I signaling complex\"\n    ],\n    \"partners\": [\n      \"MAPK1\",\n      \"PRKCЕ\",\n      \"PDLIM7\",\n      \"USP48\",\n      \"KDM6A\",\n      \"PRKG1\",\n      \"ACTA2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}