{"gene":"CAPN2","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":1991,"finding":"In transformed Schwann cells, mCANP (CAPN2) is predominantly membrane-associated (>75% activity in membrane fraction), while muCANP is predominantly cytosolic (~80%). Immunofluorescence of permeabilized cells confirmed intracellular distribution of mCANP throughout the cytoplasm with sparing of the perinuclear region.","method":"Subcellular fractionation (DEAE and phenyl Sepharose chromatography), enzyme activity assay, immunofluorescence on fixed/permeabilized vs. live cells","journal":"Journal of neuroscience research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct fractionation with activity assays and immunofluorescence, single lab, two orthogonal methods","pmids":["1656060"],"is_preprint":false},{"year":1992,"finding":"In mouse and human brain, mCANP (CAPN2) is localized predominantly within the cytosol (>95% immunoreactive content in soluble fraction after 15,000g centrifugation), with caseinolytic activity detected only in supernatant fractions; Triton X-100 extraction did not alter this distribution.","method":"Western blot immunoassay with two independent antibodies, [14C]azocasein enzymatic activity assay, differential centrifugation fractionation","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent antibodies plus enzyme activity assay, single lab","pmids":["1548485"],"is_preprint":false},{"year":2003,"finding":"The capn2 promoter contains five E-box consensus binding sites for myogenic regulatory factors and one MEF-2 binding site. MyoD transactivates capn2 via these elements and is the pivotal regulator; myogenin can upregulate capn2 with lower efficiency; Myf5 has no effect. MyoD and myogenin act synergistically on capn2 expression during myogenesis.","method":"Promoter analysis, transient transfection with reporter gene constructs, myogenic factor overexpression, antisense oligonucleotide knockdown","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assays with functional validation by antisense knockdown, single lab, multiple orthogonal methods","pmids":["12559913"],"is_preprint":false},{"year":2022,"finding":"Cisplatin activates CAPN1/CAPN2 calpain activity, which mediates pyroptosis via a CAPN1/CAPN2–BAK/BAX–caspase-9–caspase-3–GSDME signaling axis in esophageal cancer cells; calpain inhibition or knockout suppresses this cisplatin-induced pyroptosis.","method":"Western blotting, LDH release assay, calpain activity assay, cell viability assay, calpain knockout/inhibition experiments","journal":"Chemico-biological interactions","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays with genetic knockout and pharmacological inhibition, single lab","pmids":["35525317"],"is_preprint":false},{"year":2017,"finding":"Silencing CAPN2 in castration-resistant prostate cancer cells inhibits proliferation (G1 cell cycle arrest), reduces migration and invasion by decreasing MMP-2 and MMP-9 activation, and suppresses phosphorylation of AKT and mTOR, placing CAPN2 upstream of AKT/mTOR signaling.","method":"siRNA knockdown, cell cycle analysis, Transwell migration/invasion assay, MMP-2/MMP-9 activity assay, western blot for phospho-AKT and phospho-mTOR","journal":"BioMed research international","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KD with defined cellular phenotypes and pathway analysis, single lab, multiple readouts","pmids":["28280729"],"is_preprint":false},{"year":2017,"finding":"CAPN2 overexpression promotes renal cell carcinoma cell migration, invasion, and proliferation by activating AKT/mTOR signaling, enhancing epithelial-mesenchymal transition (EMT), and upregulating MMP9; sh-CAPN2 knockdown reverses these effects.","method":"sh-RNA knockdown and cDNA overexpression, Transwell assays, CCK-8 proliferation assay, western blot for AKT/mTOR signaling and EMT markers","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal gain/loss-of-function with multiple orthogonal readouts, single lab","pmids":["29228653"],"is_preprint":false},{"year":2024,"finding":"CAPN2 promotes apalutamide resistance in prostate cancer by activating protective autophagy: CAPN2 inhibits FOXO1 degradation and promotes its nuclear translocation, which transcriptionally upregulates ATG5 and thereby drives autophagy. ATF3 transcriptionally upregulates CAPN2 to reinforce this pathway.","method":"RNA sequencing, western blotting, immunofluorescence, transmission electron microscopy, nucleoplasmic protein isolation, dual-fluorescence autophagy assay","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods in single lab establishing mechanistic axis","pmids":["38844946"],"is_preprint":false},{"year":2025,"finding":"TRIM8 (an E3 ubiquitin ligase) directly interacts with CAPN2 and mediates its ubiquitination and degradation. ITLN1 binds both CAPN2 and TRIM8, enhancing CAPN2 stability by antagonizing TRIM8-mediated ubiquitination, thereby promoting ZBP1-dependent PANoptosis in intestinal epithelial cells.","method":"Co-immunoprecipitation combined with mass spectrometry, RNA-seq, rescue experiments, in vivo IL-10 KO mouse model","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS identifying ubiquitination mechanism with in vivo validation, single lab","pmids":["40520022"],"is_preprint":false},{"year":2024,"finding":"CAPN2 promotes β-catenin nuclear translocation, activating the Wnt/β-catenin pathway to inhibit mitochondrial autophagy in hepatocellular carcinoma cells; miR-124-3p directly targets the CAPN2 3'UTR to suppress CAPN2 expression and this axis is regulated by amentoflavone treatment.","method":"Dual-luciferase reporter assay, western blot, immunofluorescence (LC3), CCK-8 assay, overexpression/inhibitor experiments","journal":"Toxicology research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — luciferase reporter and western blot for mechanism, single lab, single study","pmids":["39050595"],"is_preprint":false},{"year":2023,"finding":"CAPN2 is essential for cardiac ventricle morphogenesis in Xenopus laevis; loss-of-function of calpain causes hypoplastic ventricle phenotypes in vivo, and two human CAPN2 variants (707C>T and 1112C>T) found in hypoplastic left heart syndrome patients are hypomorphic alleles.","method":"Xenopus laevis in vivo loss-of-function model, functional validation of human variants","journal":"HGG advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo vertebrate model with functional allele validation, single study","pmids":["37663545"],"is_preprint":false},{"year":2012,"finding":"Global proteolytic profiling of hepatocellular carcinoma cell lines with different metastasis potentials identified a CAPN2-centered proteolytic subnetwork including SPTBN1, ATP5B, and VIM that is more active in the highly metastatic cell line, suggesting CAPN2 cleaves these substrates to promote metastasis.","method":"PROTOMAP platform (gel-based proteomics of proteolytic fragments), bioinformatic network analysis","journal":"Proteomics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — high-throughput proteomics identifying substrates but no direct biochemical validation of individual cleavage events","pmids":["22623320"],"is_preprint":false}],"current_model":"CAPN2 (m-calpain large subunit) is a calcium-activated cysteine protease that is predominantly cytosolic in brain but membrane-associated in Schwann cells; its expression during myogenesis is transcriptionally driven by MyoD via E-box promoter elements; it functions upstream of AKT/mTOR signaling to promote cancer cell proliferation, migration, and EMT; it mediates cisplatin-induced pyroptosis via a CAPN2–BAK/BAX–caspase-9–caspase-3–GSDME axis; it promotes apalutamide resistance by stabilizing FOXO1 to transcriptionally upregulate ATG5-driven autophagy; its protein stability is regulated by TRIM8-mediated ubiquitination (opposed by ITLN1); and it is essential for vertebrate cardiac ventricle morphogenesis as demonstrated by in vivo loss-of-function in Xenopus."},"narrative":{"mechanistic_narrative":"CAPN2 (m-calpain large subunit) is a calcium-activated cysteine protease whose subcellular partitioning is cell-type-dependent: it is predominantly membrane-associated in transformed Schwann cells but almost entirely cytosolic in mouse and human brain [PMID:1656060, PMID:1548485]. During myogenesis its expression is driven transcriptionally by the myogenic regulatory factor MyoD acting through E-box elements in the capn2 promoter, with myogenin contributing synergistically [PMID:12559913]. CAPN2 acts as a pro-tumorigenic effector across multiple cancers, functioning upstream of AKT/mTOR signaling to drive proliferation, migration, invasion, MMP activation, and epithelial-mesenchymal transition [PMID:28280729, PMID:29228653]. It also controls programmed cell death and stress survival decisions through several routes: it mediates cisplatin-induced pyroptosis via a CAPN2–BAK/BAX–caspase-9–caspase-3–GSDME axis [PMID:35525317], promotes drug-resistance autophagy by stabilizing FOXO1 to transcriptionally upregulate ATG5 [PMID:38844946], and supports ZBP1-dependent PANoptosis in intestinal epithelium [PMID:40520022]. CAPN2 protein stability is set by TRIM8-mediated ubiquitination and degradation, which is antagonized by ITLN1 [PMID:40520022]. In vivo, CAPN2 is required for vertebrate cardiac ventricle morphogenesis, and hypomorphic human variants are found in hypoplastic left heart syndrome patients [PMID:37663545].","teleology":[{"year":1992,"claim":"Resolved where m-calpain resides in tissue, establishing that CAPN2 subcellular localization is context-dependent rather than fixed — membrane-associated in transformed Schwann cells but cytosolic in brain.","evidence":"Subcellular fractionation with activity assays and immunofluorescence in Schwann cells, and differential centrifugation with two antibodies plus enzyme assay in brain","pmids":["1656060","1548485"],"confidence":"Medium","gaps":["Determinants of membrane vs cytosolic partitioning not defined","No structural basis for the localization difference","Functional consequence of each pool not established"]},{"year":2003,"claim":"Identified how CAPN2 is transcriptionally controlled during muscle differentiation, placing it within the MyoD-driven myogenic program.","evidence":"Promoter reporter assays with myogenic factor overexpression and antisense knockdown","pmids":["12559913"],"confidence":"Medium","gaps":["Direct MyoD occupancy at endogenous capn2 promoter not shown","Physiological role of CAPN2 in myogenesis not functionally dissected"]},{"year":2012,"claim":"Sought CAPN2 substrates relevant to metastasis, nominating a proteolytic subnetwork (SPTBN1, ATP5B, VIM) in hepatocellular carcinoma.","evidence":"PROTOMAP gel-based proteolytic-fragment proteomics with network analysis","pmids":["22623320"],"confidence":"Low","gaps":["No direct biochemical validation of individual cleavage events","Substrate cleavage sites not mapped","Causal link between cleavage and metastasis not tested"]},{"year":2017,"claim":"Established CAPN2 as a driver of cancer cell proliferation and invasion acting upstream of AKT/mTOR, connecting protease activity to a defined oncogenic signaling output.","evidence":"Reciprocal siRNA/shRNA knockdown and overexpression with cell cycle, Transwell, MMP and phospho-AKT/mTOR readouts in prostate and renal carcinoma cells","pmids":["28280729","29228653"],"confidence":"Medium","gaps":["Direct proteolytic substrate linking CAPN2 to AKT/mTOR not identified","Whether the effect requires catalytic activity not tested","In vivo tumor relevance limited"]},{"year":2022,"claim":"Defined a cell-death role for CAPN2 in chemotherapy response, placing it at the head of a pyroptotic cascade.","evidence":"Calpain activity assays, LDH release, and knockout/inhibition in cisplatin-treated esophageal cancer cells","pmids":["35525317"],"confidence":"Medium","gaps":["Direct CAPN2 substrate in the BAK/BAX step not identified","CAPN1 vs CAPN2 specific contributions not separated"]},{"year":2024,"claim":"Revealed CAPN2-driven protective autophagy as a mechanism of androgen-pathway drug resistance via FOXO1 stabilization and ATG5 induction.","evidence":"RNA-seq, nucleoplasmic fractionation, TEM, and dual-fluorescence autophagy assays in apalutamide-resistant prostate cancer cells","pmids":["38844946"],"confidence":"Medium","gaps":["Whether FOXO1 stabilization is via direct CAPN2 proteolysis unclear","ATF3→CAPN2 regulation mechanism not detailed"]},{"year":2024,"claim":"Linked CAPN2 to Wnt/β-catenin-mediated suppression of mitophagy and identified miR-124-3p as an upstream repressor.","evidence":"Dual-luciferase reporter, LC3 immunofluorescence and western blot in hepatocellular carcinoma cells","pmids":["39050595"],"confidence":"Low","gaps":["Single study, mechanism rests on reporter and western blot only","Direct CAPN2–β-catenin interaction not shown","Catalytic requirement not tested"]},{"year":2025,"claim":"Identified how CAPN2 protein abundance is post-translationally controlled, defining a TRIM8–ITLN1 axis governing its stability and downstream PANoptosis.","evidence":"Co-IP/MS, rescue experiments and an IL-10 KO mouse model in intestinal epithelial cells","pmids":["40520022"],"confidence":"Medium","gaps":["TRIM8 ubiquitination sites on CAPN2 not mapped","How CAPN2 mechanistically promotes ZBP1-dependent PANoptosis not resolved"]},{"year":2023,"claim":"Demonstrated an in vivo developmental requirement for CAPN2 in heart formation and tied human hypomorphic alleles to congenital heart disease.","evidence":"Xenopus laevis loss-of-function model with functional validation of two human CAPN2 variants","pmids":["37663545"],"confidence":"Medium","gaps":["Molecular substrates relevant to ventricle morphogenesis unknown","Variant functional assays limited to model system"]},{"year":null,"claim":"The direct proteolytic substrates that mechanistically connect CAPN2 to AKT/mTOR signaling, cell-death cascades, and cardiac morphogenesis remain largely undefined.","evidence":"","pmids":[],"confidence":"Low","gaps":["No validated catalytic substrate links CAPN2 activity to its phenotypic outputs","Structural basis of activation and localization unknown","Whether oncogenic roles require catalytic activity untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,10]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,7]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[6,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,5]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[9]}],"complexes":[],"partners":["TRIM8","ITLN1","FOXO1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P17655","full_name":"Calpain-2 catalytic subunit","aliases":["Calcium-activated neutral proteinase 2","CANP 2","Calpain M-type","Calpain large polypeptide L2","Calpain-2 large subunit","Millimolar-calpain","M-calpain"],"length_aa":700,"mass_kda":80.0,"function":"Calcium-regulated non-lysosomal thiol-protease which catalyzes limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction. Proteolytically cleaves MYOC at 'Arg-226' (PubMed:17650508). Proteolytically cleaves CPEB3 following neuronal stimulation which abolishes CPEB3 translational repressor activity, leading to translation of CPEB3 target mRNAs (By similarity)","subcellular_location":"Cytoplasm; Cell membrane","url":"https://www.uniprot.org/uniprotkb/P17655/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CAPN2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CAPN2","total_profiled":1310},"omim":[{"mim_id":"618777","title":"CALPAIN 8; CAPN8","url":"https://www.omim.org/entry/618777"},{"mim_id":"616874","title":"TRANSMEMBRANE BAX INHIBITOR MOTIF-CONTAINING PROTEIN 4; TMBIM4","url":"https://www.omim.org/entry/616874"},{"mim_id":"616767","title":"CALPAIN, SMALL SUBUNIT 2; CAPNS2","url":"https://www.omim.org/entry/616767"},{"mim_id":"608839","title":"CALPAIN 12; CAPN12","url":"https://www.omim.org/entry/608839"},{"mim_id":"606401","title":"CALPAIN 9; CAPN9","url":"https://www.omim.org/entry/606401"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Connecting piece","reliability":"Supported"},{"location":"Mid piece","reliability":"Supported"},{"location":"Principal piece","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CAPN2"},"hgnc":{"alias_symbol":["mCANP","CANPml","CANPL2"],"prev_symbol":[]},"alphafold":{"accession":"P17655","domains":[{"cath_id":"-","chopping":"25-204","consensus_level":"high","plddt":94.423,"start":25,"end":204},{"cath_id":"3.90.70.10","chopping":"212-310_319-343","consensus_level":"high","plddt":90.8219,"start":212,"end":343},{"cath_id":"2.60.120.380","chopping":"357-393_407-418_429-436_460-513","consensus_level":"high","plddt":90.5665,"start":357,"end":513},{"cath_id":"1.10.238.10","chopping":"528-700","consensus_level":"high","plddt":87.9303,"start":528,"end":700}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P17655","model_url":"https://alphafold.ebi.ac.uk/files/AF-P17655-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P17655-F1-predicted_aligned_error_v6.png","plddt_mean":89.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CAPN2","jax_strain_url":"https://www.jax.org/strain/search?query=CAPN2"},"sequence":{"accession":"P17655","fasta_url":"https://rest.uniprot.org/uniprotkb/P17655.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P17655/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P17655"}},"corpus_meta":[{"pmid":"15621511","id":"PMC_15621511","title":"Identification and molecular characterization of the rainbow trout calpains (Capn1 and Capn2): their expression in muscle wasting during starvation.","date":"2005","source":"Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15621511","citation_count":47,"is_preprint":false},{"pmid":"35525317","id":"PMC_35525317","title":"Cisplatin-induced pyroptosis is mediated via the CAPN1/CAPN2-BAK/BAX-caspase-9-caspase-3-GSDME axis in esophageal cancer.","date":"2022","source":"Chemico-biological interactions","url":"https://pubmed.ncbi.nlm.nih.gov/35525317","citation_count":45,"is_preprint":false},{"pmid":"33731671","id":"PMC_33731671","title":"hnRNPK-regulated LINC00263 promotes malignant phenotypes through miR-147a/CAPN2.","date":"2021","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/33731671","citation_count":32,"is_preprint":false},{"pmid":"1656060","id":"PMC_1656060","title":"Calcium-activated neutral proteinase (CANP; calpain) activity in Schwann cells: immunofluorescence localization and compartmentation of mu- and mCANP.","date":"1991","source":"Journal of neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/1656060","citation_count":24,"is_preprint":false},{"pmid":"28280729","id":"PMC_28280729","title":"Silencing CAPN2 Expression Inhibited Castration-Resistant Prostate Cancer Cells Proliferation and Invasion via AKT/mTOR Signal Pathway.","date":"2017","source":"BioMed research international","url":"https://pubmed.ncbi.nlm.nih.gov/28280729","citation_count":23,"is_preprint":false},{"pmid":"1548485","id":"PMC_1548485","title":"Immunoassay and activity of calcium-activated neutral proteinase (mCANP): distribution in soluble and membrane-associated fractions in human and mouse brain.","date":"1992","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1548485","citation_count":22,"is_preprint":false},{"pmid":"29228653","id":"PMC_29228653","title":"Overexpression of CAPN2 promotes cell metastasis and proliferation via AKT/mTOR signaling in renal cell carcinoma.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29228653","citation_count":21,"is_preprint":false},{"pmid":"22623320","id":"PMC_22623320","title":"Global profiling of proteolytically modified proteins in human metastatic hepatocellular carcinoma cell lines reveals CAPN2 centered network.","date":"2012","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/22623320","citation_count":21,"is_preprint":false},{"pmid":"20093171","id":"PMC_20093171","title":"Characterisation of capn1, capn2-like, capn3 and capn11 genes in Atlantic halibut (Hippoglossus hippoglossus L.): Transcriptional regulation across tissues and in skeletal muscle at distinct nutritional states.","date":"2010","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/20093171","citation_count":18,"is_preprint":false},{"pmid":"12559913","id":"PMC_12559913","title":"Transactivation of capn2 by myogenic regulatory factors during myogenesis.","date":"2003","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12559913","citation_count":17,"is_preprint":false},{"pmid":"32951272","id":"PMC_32951272","title":"Promotive effect of Talin-1 protein on gastric cancer progression through PTK2-PXN-VCL-E-Cadherin-CAPN2-MAPK1 signaling axis.","date":"2020","source":"Journal of clinical laboratory analysis","url":"https://pubmed.ncbi.nlm.nih.gov/32951272","citation_count":17,"is_preprint":false},{"pmid":"32382656","id":"PMC_32382656","title":"miR-124 Intensified Oxaliplatin-Based Chemotherapy by Targeting CAPN2 in Colorectal Cancer.","date":"2020","source":"Molecular therapy oncolytics","url":"https://pubmed.ncbi.nlm.nih.gov/32382656","citation_count":15,"is_preprint":false},{"pmid":"40520022","id":"PMC_40520022","title":"ITLN1 exacerbates Crohn's colitis by driving ZBP1-dependent PANoptosis in intestinal epithelial cells through antagonizing TRIM8-mediated CAPN2 ubiquitination.","date":"2025","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40520022","citation_count":13,"is_preprint":false},{"pmid":"28002826","id":"PMC_28002826","title":"The CAPN2/CAPN8 Locus on Chromosome 1q Is Associated with Variation in Serum Alpha-Carotene Concentrations.","date":"2016","source":"Journal of nutrigenetics and nutrigenomics","url":"https://pubmed.ncbi.nlm.nih.gov/28002826","citation_count":10,"is_preprint":false},{"pmid":"39434688","id":"PMC_39434688","title":"METTL16 inhibits pancreatic cancer proliferation and metastasis by promoting MROH8 RNA stability and inhibiting CAPN2 expression - experimental studies.","date":"2024","source":"International journal of surgery (London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/39434688","citation_count":8,"is_preprint":false},{"pmid":"31680308","id":"PMC_31680308","title":"CAPN2 acts as an indicator of hepatitis B virus to induce hepatic fibrosis.","date":"2019","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/31680308","citation_count":8,"is_preprint":false},{"pmid":"38844946","id":"PMC_38844946","title":"CAPN2 promotes apalutamide resistance in metastatic hormone-sensitive prostate cancer by activating protective autophagy.","date":"2024","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38844946","citation_count":7,"is_preprint":false},{"pmid":"38610028","id":"PMC_38610028","title":"CAPN2 correlates with insulin resistance states in PCOS as evidenced by multi-dataset analysis.","date":"2024","source":"Journal of ovarian research","url":"https://pubmed.ncbi.nlm.nih.gov/38610028","citation_count":4,"is_preprint":false},{"pmid":"37663545","id":"PMC_37663545","title":"Rare variants in CAPN2 increase risk for isolated hypoplastic left heart syndrome.","date":"2023","source":"HGG advances","url":"https://pubmed.ncbi.nlm.nih.gov/37663545","citation_count":3,"is_preprint":false},{"pmid":"39050595","id":"PMC_39050595","title":"Amentoflavone regulates the miR-124-3p/CAPN2 axis to promote mitochondrial autophagy in HCC cells.","date":"2024","source":"Toxicology research","url":"https://pubmed.ncbi.nlm.nih.gov/39050595","citation_count":3,"is_preprint":false},{"pmid":"21961413","id":"PMC_21961413","title":"[Beta-carotene regulates the expression of proapoptotic BAX and CAPN2 in HL-60, U-937 and TF-1 - human acute myeloid leukemia cell lines; microarray, RQ-PCR and Western Blot analysis].","date":"2011","source":"Przeglad lekarski","url":"https://pubmed.ncbi.nlm.nih.gov/21961413","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13684,"output_tokens":2727,"usd":0.040979,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10018,"output_tokens":3177,"usd":0.064757,"stage2_stop_reason":"end_turn"},"total_usd":0.105736,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"In transformed Schwann cells, mCANP (CAPN2) is predominantly membrane-associated (>75% activity in membrane fraction), while muCANP is predominantly cytosolic (~80%). Immunofluorescence of permeabilized cells confirmed intracellular distribution of mCANP throughout the cytoplasm with sparing of the perinuclear region.\",\n      \"method\": \"Subcellular fractionation (DEAE and phenyl Sepharose chromatography), enzyme activity assay, immunofluorescence on fixed/permeabilized vs. live cells\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct fractionation with activity assays and immunofluorescence, single lab, two orthogonal methods\",\n      \"pmids\": [\"1656060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"In mouse and human brain, mCANP (CAPN2) is localized predominantly within the cytosol (>95% immunoreactive content in soluble fraction after 15,000g centrifugation), with caseinolytic activity detected only in supernatant fractions; Triton X-100 extraction did not alter this distribution.\",\n      \"method\": \"Western blot immunoassay with two independent antibodies, [14C]azocasein enzymatic activity assay, differential centrifugation fractionation\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent antibodies plus enzyme activity assay, single lab\",\n      \"pmids\": [\"1548485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The capn2 promoter contains five E-box consensus binding sites for myogenic regulatory factors and one MEF-2 binding site. MyoD transactivates capn2 via these elements and is the pivotal regulator; myogenin can upregulate capn2 with lower efficiency; Myf5 has no effect. MyoD and myogenin act synergistically on capn2 expression during myogenesis.\",\n      \"method\": \"Promoter analysis, transient transfection with reporter gene constructs, myogenic factor overexpression, antisense oligonucleotide knockdown\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assays with functional validation by antisense knockdown, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"12559913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cisplatin activates CAPN1/CAPN2 calpain activity, which mediates pyroptosis via a CAPN1/CAPN2–BAK/BAX–caspase-9–caspase-3–GSDME signaling axis in esophageal cancer cells; calpain inhibition or knockout suppresses this cisplatin-induced pyroptosis.\",\n      \"method\": \"Western blotting, LDH release assay, calpain activity assay, cell viability assay, calpain knockout/inhibition experiments\",\n      \"journal\": \"Chemico-biological interactions\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays with genetic knockout and pharmacological inhibition, single lab\",\n      \"pmids\": [\"35525317\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Silencing CAPN2 in castration-resistant prostate cancer cells inhibits proliferation (G1 cell cycle arrest), reduces migration and invasion by decreasing MMP-2 and MMP-9 activation, and suppresses phosphorylation of AKT and mTOR, placing CAPN2 upstream of AKT/mTOR signaling.\",\n      \"method\": \"siRNA knockdown, cell cycle analysis, Transwell migration/invasion assay, MMP-2/MMP-9 activity assay, western blot for phospho-AKT and phospho-mTOR\",\n      \"journal\": \"BioMed research international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KD with defined cellular phenotypes and pathway analysis, single lab, multiple readouts\",\n      \"pmids\": [\"28280729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CAPN2 overexpression promotes renal cell carcinoma cell migration, invasion, and proliferation by activating AKT/mTOR signaling, enhancing epithelial-mesenchymal transition (EMT), and upregulating MMP9; sh-CAPN2 knockdown reverses these effects.\",\n      \"method\": \"sh-RNA knockdown and cDNA overexpression, Transwell assays, CCK-8 proliferation assay, western blot for AKT/mTOR signaling and EMT markers\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal gain/loss-of-function with multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"29228653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CAPN2 promotes apalutamide resistance in prostate cancer by activating protective autophagy: CAPN2 inhibits FOXO1 degradation and promotes its nuclear translocation, which transcriptionally upregulates ATG5 and thereby drives autophagy. ATF3 transcriptionally upregulates CAPN2 to reinforce this pathway.\",\n      \"method\": \"RNA sequencing, western blotting, immunofluorescence, transmission electron microscopy, nucleoplasmic protein isolation, dual-fluorescence autophagy assay\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods in single lab establishing mechanistic axis\",\n      \"pmids\": [\"38844946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRIM8 (an E3 ubiquitin ligase) directly interacts with CAPN2 and mediates its ubiquitination and degradation. ITLN1 binds both CAPN2 and TRIM8, enhancing CAPN2 stability by antagonizing TRIM8-mediated ubiquitination, thereby promoting ZBP1-dependent PANoptosis in intestinal epithelial cells.\",\n      \"method\": \"Co-immunoprecipitation combined with mass spectrometry, RNA-seq, rescue experiments, in vivo IL-10 KO mouse model\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS identifying ubiquitination mechanism with in vivo validation, single lab\",\n      \"pmids\": [\"40520022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CAPN2 promotes β-catenin nuclear translocation, activating the Wnt/β-catenin pathway to inhibit mitochondrial autophagy in hepatocellular carcinoma cells; miR-124-3p directly targets the CAPN2 3'UTR to suppress CAPN2 expression and this axis is regulated by amentoflavone treatment.\",\n      \"method\": \"Dual-luciferase reporter assay, western blot, immunofluorescence (LC3), CCK-8 assay, overexpression/inhibitor experiments\",\n      \"journal\": \"Toxicology research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — luciferase reporter and western blot for mechanism, single lab, single study\",\n      \"pmids\": [\"39050595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CAPN2 is essential for cardiac ventricle morphogenesis in Xenopus laevis; loss-of-function of calpain causes hypoplastic ventricle phenotypes in vivo, and two human CAPN2 variants (707C>T and 1112C>T) found in hypoplastic left heart syndrome patients are hypomorphic alleles.\",\n      \"method\": \"Xenopus laevis in vivo loss-of-function model, functional validation of human variants\",\n      \"journal\": \"HGG advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo vertebrate model with functional allele validation, single study\",\n      \"pmids\": [\"37663545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Global proteolytic profiling of hepatocellular carcinoma cell lines with different metastasis potentials identified a CAPN2-centered proteolytic subnetwork including SPTBN1, ATP5B, and VIM that is more active in the highly metastatic cell line, suggesting CAPN2 cleaves these substrates to promote metastasis.\",\n      \"method\": \"PROTOMAP platform (gel-based proteomics of proteolytic fragments), bioinformatic network analysis\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — high-throughput proteomics identifying substrates but no direct biochemical validation of individual cleavage events\",\n      \"pmids\": [\"22623320\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CAPN2 (m-calpain large subunit) is a calcium-activated cysteine protease that is predominantly cytosolic in brain but membrane-associated in Schwann cells; its expression during myogenesis is transcriptionally driven by MyoD via E-box promoter elements; it functions upstream of AKT/mTOR signaling to promote cancer cell proliferation, migration, and EMT; it mediates cisplatin-induced pyroptosis via a CAPN2–BAK/BAX–caspase-9–caspase-3–GSDME axis; it promotes apalutamide resistance by stabilizing FOXO1 to transcriptionally upregulate ATG5-driven autophagy; its protein stability is regulated by TRIM8-mediated ubiquitination (opposed by ITLN1); and it is essential for vertebrate cardiac ventricle morphogenesis as demonstrated by in vivo loss-of-function in Xenopus.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CAPN2 (m-calpain large subunit) is a calcium-activated cysteine protease whose subcellular partitioning is cell-type-dependent: it is predominantly membrane-associated in transformed Schwann cells but almost entirely cytosolic in mouse and human brain [#0, #1]. During myogenesis its expression is driven transcriptionally by the myogenic regulatory factor MyoD acting through E-box elements in the capn2 promoter, with myogenin contributing synergistically [#2]. CAPN2 acts as a pro-tumorigenic effector across multiple cancers, functioning upstream of AKT/mTOR signaling to drive proliferation, migration, invasion, MMP activation, and epithelial-mesenchymal transition [#4, #5]. It also controls programmed cell death and stress survival decisions through several routes: it mediates cisplatin-induced pyroptosis via a CAPN2\\u2013BAK/BAX\\u2013caspase-9\\u2013caspase-3\\u2013GSDME axis [#3], promotes drug-resistance autophagy by stabilizing FOXO1 to transcriptionally upregulate ATG5 [#6], and supports ZBP1-dependent PANoptosis in intestinal epithelium [#7]. CAPN2 protein stability is set by TRIM8-mediated ubiquitination and degradation, which is antagonized by ITLN1 [#7]. In vivo, CAPN2 is required for vertebrate cardiac ventricle morphogenesis, and hypomorphic human variants are found in hypoplastic left heart syndrome patients [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Resolved where m-calpain resides in tissue, establishing that CAPN2 subcellular localization is context-dependent rather than fixed \\u2014 membrane-associated in transformed Schwann cells but cytosolic in brain.\",\n      \"evidence\": \"Subcellular fractionation with activity assays and immunofluorescence in Schwann cells, and differential centrifugation with two antibodies plus enzyme assay in brain\",\n      \"pmids\": [\"1656060\", \"1548485\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Determinants of membrane vs cytosolic partitioning not defined\", \"No structural basis for the localization difference\", \"Functional consequence of each pool not established\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified how CAPN2 is transcriptionally controlled during muscle differentiation, placing it within the MyoD-driven myogenic program.\",\n      \"evidence\": \"Promoter reporter assays with myogenic factor overexpression and antisense knockdown\",\n      \"pmids\": [\"12559913\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct MyoD occupancy at endogenous capn2 promoter not shown\", \"Physiological role of CAPN2 in myogenesis not functionally dissected\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Sought CAPN2 substrates relevant to metastasis, nominating a proteolytic subnetwork (SPTBN1, ATP5B, VIM) in hepatocellular carcinoma.\",\n      \"evidence\": \"PROTOMAP gel-based proteolytic-fragment proteomics with network analysis\",\n      \"pmids\": [\"22623320\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical validation of individual cleavage events\", \"Substrate cleavage sites not mapped\", \"Causal link between cleavage and metastasis not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established CAPN2 as a driver of cancer cell proliferation and invasion acting upstream of AKT/mTOR, connecting protease activity to a defined oncogenic signaling output.\",\n      \"evidence\": \"Reciprocal siRNA/shRNA knockdown and overexpression with cell cycle, Transwell, MMP and phospho-AKT/mTOR readouts in prostate and renal carcinoma cells\",\n      \"pmids\": [\"28280729\", \"29228653\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct proteolytic substrate linking CAPN2 to AKT/mTOR not identified\", \"Whether the effect requires catalytic activity not tested\", \"In vivo tumor relevance limited\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined a cell-death role for CAPN2 in chemotherapy response, placing it at the head of a pyroptotic cascade.\",\n      \"evidence\": \"Calpain activity assays, LDH release, and knockout/inhibition in cisplatin-treated esophageal cancer cells\",\n      \"pmids\": [\"35525317\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CAPN2 substrate in the BAK/BAX step not identified\", \"CAPN1 vs CAPN2 specific contributions not separated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed CAPN2-driven protective autophagy as a mechanism of androgen-pathway drug resistance via FOXO1 stabilization and ATG5 induction.\",\n      \"evidence\": \"RNA-seq, nucleoplasmic fractionation, TEM, and dual-fluorescence autophagy assays in apalutamide-resistant prostate cancer cells\",\n      \"pmids\": [\"38844946\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether FOXO1 stabilization is via direct CAPN2 proteolysis unclear\", \"ATF3\\u2192CAPN2 regulation mechanism not detailed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked CAPN2 to Wnt/\\u03b2-catenin-mediated suppression of mitophagy and identified miR-124-3p as an upstream repressor.\",\n      \"evidence\": \"Dual-luciferase reporter, LC3 immunofluorescence and western blot in hepatocellular carcinoma cells\",\n      \"pmids\": [\"39050595\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single study, mechanism rests on reporter and western blot only\", \"Direct CAPN2\\u2013\\u03b2-catenin interaction not shown\", \"Catalytic requirement not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified how CAPN2 protein abundance is post-translationally controlled, defining a TRIM8\\u2013ITLN1 axis governing its stability and downstream PANoptosis.\",\n      \"evidence\": \"Co-IP/MS, rescue experiments and an IL-10 KO mouse model in intestinal epithelial cells\",\n      \"pmids\": [\"40520022\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TRIM8 ubiquitination sites on CAPN2 not mapped\", \"How CAPN2 mechanistically promotes ZBP1-dependent PANoptosis not resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated an in vivo developmental requirement for CAPN2 in heart formation and tied human hypomorphic alleles to congenital heart disease.\",\n      \"evidence\": \"Xenopus laevis loss-of-function model with functional validation of two human CAPN2 variants\",\n      \"pmids\": [\"37663545\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrates relevant to ventricle morphogenesis unknown\", \"Variant functional assays limited to model system\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct proteolytic substrates that mechanistically connect CAPN2 to AKT/mTOR signaling, cell-death cascades, and cardiac morphogenesis remain largely undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No validated catalytic substrate links CAPN2 activity to its phenotypic outputs\", \"Structural basis of activation and localization unknown\", \"Whether oncogenic roles require catalytic activity untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 10]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 7]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TRIM8\", \"ITLN1\", \"FOXO1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}