{"gene":"CAPN7","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2008,"finding":"CAPN7 (calpain 7/PalBH) interacts with a subset of ESCRT-III-related proteins (CHMPs) through its N-terminal tandem MIT domains; direct interaction with CHMP1B was confirmed by pulldown using recombinant proteins. Endogenous CAPN7 is mostly cytosolic but a fraction localizes to particulate/endosomal compartments, and overexpression of dominant-negative VPS4B causes CAPN7 accumulation in perinuclear puncta that partly co-localize with endocytosed EGF.","method":"Pulldown assay (Strep-tagged stable transfectants and recombinant proteins), subcellular fractionation, fluorescence microscopy, GFP-EGF co-localization","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1-2 — direct interaction confirmed with recombinant proteins, multiple orthogonal methods (pulldown, fractionation, live imaging), foundational study","pmids":["18316332"],"is_preprint":false},{"year":2014,"finding":"CAPN7 proteolytic activity is required for efficient EGFR degradation via the endosomal sorting pathway. MIT-domain-dependent interaction with ESCRT proteins (including IST1) recruits CAPN7 to EGFR-positive endosomes upon EGF stimulation; a protease-inactive mutant (C290S) acts as dominant-negative and delays EGFR degradation, an effect abolished by deletion of the MIT domains. IST1 knockdown prevents CAPN7(C290S) co-localization with EGFR.","method":"CAPN7-knockdown HeLa cells and Capn7-/- MEFs, re-expression of wild-type vs. C290S mutant, MIT domain deletion mutants, immunofluorescence microscopy, EGF-stimulated EGFR degradation assay","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1-2 — KO cells + rescue with active-site mutant and domain deletion mutants, multiple orthogonal readouts","pmids":["24953135"],"is_preprint":false},{"year":2018,"finding":"CAPN7 degrades HOXA10 in a Ca2+-dependent manner via a PEST sequence in HOXA10; overexpression of CAPN7 in Ishikawa cells or mouse uterus inhibits embryo implantation. Interaction between HOXA10 and CAPN7 represses HOXA10 transcriptional activity and protein stability. Truncation of the PEST motif in HOXA10 abolishes CAPN7-dependent proteolysis; the calpain inhibitor ALLN reverses CAPN7-induced HOXA10 degradation.","method":"Overexpression in Ishikawa cells and in vivo mouse uterus model, PEST-motif truncation mutants, calpain inhibitor (ALLN) rescue, in vitro Ca2+-dependent degradation assay, co-immunoprecipitation","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro degradation assay, domain truncation mutants, in vivo rescue with inhibitor, multiple orthogonal methods","pmids":["29459744"],"is_preprint":false},{"year":2019,"finding":"CAPN7 mediates protease-dependent degradation of SMN (survival motor neuron) protein; the cysteine protease inhibitor Z-FA-FMK stabilizes SMN by blocking CAPN7 (among CAPN1, CTSB, and CTSL) activity, identifying CAPN7 as one of the proteases responsible for SMN turnover.","method":"SMN2-GFP reporter drug screen, cysteine protease inhibitor treatment, Western blot analysis of SMN levels, identification of CAPN7 as mediator of SMN degradation","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 3 — pharmacological inhibitor approach without direct substrate confirmation or mutagenesis specific to CAPN7; CAPN7 identified as one of four proteases","pmids":["30910806"],"is_preprint":false},{"year":2022,"finding":"CAPN7 hydrolyzes AKT1 (which contains a conserved PEST sequence) and enhances AKT1 phosphorylation, thereby promoting phosphorylation of the downstream transcription factor FoxO1 at Ser319 and increasing FoxO1 nuclear exclusion, which attenuates FoxO1 transcriptional activity and negatively regulates decidualization of human endometrial stromal cells.","method":"CAPN7 overexpression/knockdown in HESCs, in vitro and in vivo decidualization models, PEST sequence identification in AKT1, co-immunoprecipitation, phospho-specific Western blotting, nuclear/cytoplasmic fractionation","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2-3 — PEST-dependent cleavage supported by IP and phospho-blots, in vivo phenotype, but reconstituted in vitro cleavage not fully reported","pmids":["35191464"],"is_preprint":false},{"year":2022,"finding":"CAPN7 localizes to cytokinetic midbody membrane bridges through interaction with its specific ESCRT-III binding partner IST1, and is required for both efficient cytokinetic abscission and maintenance of the NoCut abscission checkpoint. Quantitative mapping of MIT–ESCRT-III interactions showed the CAPN7 MIT domain binds IST1 among 228 pairwise interactions tested.","method":"Comprehensive quantitative MIT–ESCRT-III interaction mapping (228 pairwise), live-cell imaging of cytokinetic midbodies, CAPN7 depletion/rescue abscission assays, NoCut checkpoint assays, crystal structure of SPASTIN-IST1 (as part of same study)","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1-2 — comprehensive quantitative interactome, functional abscission assays, checkpoint assays, replicated across multiple MIT enzymes in same study","pmids":["36107470"],"is_preprint":false},{"year":2023,"finding":"The tandem MIT domains of CAPN7 bind simultaneously to two distinct MIT-interaction motifs (MIMs) of IST1; crystal structures of the CAPN7 tandem-MIT–IST1 complex were solved, and structure-guided point mutations in either MIT domain disrupted IST1 binding in vitro and in cells. Depletion/rescue experiments demonstrated that the CAPN7–IST1 interaction is required for (1) CAPN7 recruitment to midbodies, (2) efficient abscission, and (3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also independently required for abscission and checkpoint maintenance.","method":"Crystallography (tandem MIT–IST1 complex), structure-guided mutagenesis, in vitro binding assays, CAPN7 depletion/rescue in cells, abscission timing assays, NoCut checkpoint assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis plus functional cellular rescue with multiple orthogonal readouts","pmids":["37772788"],"is_preprint":false},{"year":2019,"finding":"CAPN7 lacks a penta-EF-hand domain but contains two tandem MIT domains that interact with a subset of ESCRT-III proteins to regulate EGF receptor downregulation, distinguishing its mechanism from classical calpains.","method":"Review/synthesis of structural and functional data including MIT domain interaction studies and EGFR degradation experiments","journal":"Bioscience, biotechnology, and biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 — review article synthesizing prior experimental work; no new primary experiments","pmids":["31814542"],"is_preprint":false}],"current_model":"CAPN7 is an atypical cysteine protease that lacks a penta-EF-hand domain but contains tandem MIT domains, which directly bind ESCRT-III subunits (principally IST1, via two distinct MIMs simultaneously) to recruit CAPN7 to endosomes and the cytokinetic midbody; there, its proteolytic activity is required for EGFR degradation via the endosomal sorting pathway and for both efficient cytokinetic abscission and NoCut checkpoint maintenance, and it can also cleave substrates containing PEST sequences (including HOXA10 and AKT1) to modulate transcription factor stability and signaling."},"narrative":{"teleology":[{"year":2008,"claim":"Establishing that CAPN7 physically engages the ESCRT-III machinery resolved how an atypical calpain without a penta-EF-hand domain could be targeted to endosomal membranes.","evidence":"Recombinant pulldown confirmed direct CAPN7–CHMP1B interaction via tandem MIT domains; subcellular fractionation and fluorescence microscopy showed partial endosomal localization enhanced by dominant-negative VPS4B in HeLa cells.","pmids":["18316332"],"confidence":"High","gaps":["Proteolytic activity was not tested; whether ESCRT-III binding serves a catalytic or scaffolding purpose remained open.","The preferred ESCRT-III partner(s) beyond CHMP1B were not ranked quantitatively.","Endogenous substrate(s) at endosomes were unknown."]},{"year":2014,"claim":"Demonstrating that CAPN7 protease activity is required for EGFR degradation linked the MIT-domain-mediated ESCRT-III recruitment to a specific endosomal sorting function.","evidence":"CAPN7 knockdown and Capn7−/− MEFs showed delayed EGF-stimulated EGFR degradation; rescue with catalytic-dead C290S mutant acted dominant-negatively, an effect abolished by MIT domain deletion; IST1 knockdown prevented CAPN7(C290S) co-localization with EGFR in HeLa cells.","pmids":["24953135"],"confidence":"High","gaps":["The direct endosomal substrate(s) cleaved by CAPN7 to facilitate EGFR degradation were not identified.","Whether CAPN7 acts on EGFR itself or on ESCRT-associated factors was unresolved."]},{"year":2018,"claim":"Identifying HOXA10 as a direct PEST-dependent substrate expanded CAPN7 function beyond endosomal sorting to transcription factor turnover and reproductive biology.","evidence":"In vitro Ca²⁺-dependent degradation assay, PEST-motif truncation abolished cleavage, calpain inhibitor ALLN rescued HOXA10 stability; CAPN7 overexpression in mouse uterus inhibited embryo implantation.","pmids":["29459744"],"confidence":"High","gaps":["The precise cleavage site(s) within HOXA10 were not mapped.","Whether CAPN7-mediated HOXA10 degradation operates via endosomal or cytosolic pools was unclear."]},{"year":2022,"claim":"Systematic MIT–ESCRT-III interaction mapping identified IST1 as the principal CAPN7 binding partner and showed that this interaction recruits CAPN7 to the cytokinetic midbody for abscission and NoCut checkpoint control, revealing a second major cellular function beyond endosomal sorting.","evidence":"Quantitative pairwise mapping of 228 MIT–ESCRT-III interactions, live-cell abscission timing assays, and NoCut checkpoint assays upon CAPN7 depletion/rescue.","pmids":["36107470"],"confidence":"High","gaps":["The midbody substrate(s) of CAPN7 protease activity were not identified.","Whether the abscission and checkpoint functions require the same or different proteolytic events was unresolved."]},{"year":2022,"claim":"Demonstrating that CAPN7 cleaves the PEST-containing kinase AKT1 and modulates FoxO1 phosphorylation extended the substrate repertoire to a major signaling node controlling endometrial decidualization.","evidence":"CAPN7 overexpression/knockdown in human endometrial stromal cells, co-immunoprecipitation with AKT1, phospho-specific Western blotting for FoxO1, nuclear/cytoplasmic fractionation.","pmids":["35191464"],"confidence":"Medium","gaps":["Reconstituted in vitro cleavage of AKT1 by purified CAPN7 was not fully demonstrated.","The precise AKT1 cleavage site and whether this generates a constitutively active fragment were not determined.","The relationship between AKT1 hydrolysis and enhanced AKT1 phosphorylation is mechanistically unclear."]},{"year":2023,"claim":"Crystal structures of the CAPN7 tandem-MIT–IST1 complex revealed a bipartite binding mode in which both MIT domains simultaneously engage two MIMs on IST1, providing the structural basis for CAPN7 recruitment to midbodies and its roles in abscission and the NoCut checkpoint.","evidence":"X-ray crystallography of tandem-MIT–IST1 complex, structure-guided point mutations disrupting IST1 binding in vitro and in cells, depletion/rescue abscission and checkpoint assays demonstrating that both MIT-mediated recruitment and catalytic activity are independently required.","pmids":["37772788"],"confidence":"High","gaps":["The midbody substrate(s) whose cleavage mediates abscission timing and checkpoint signaling remain unidentified.","How catalytic activity and IST1-mediated recruitment are coordinated is not structurally resolved.","Whether additional ESCRT-III subunits contribute to CAPN7 regulation at the midbody in vivo is unknown."]},{"year":null,"claim":"The direct proteolytic substrates of CAPN7 at endosomes and at the cytokinetic midbody remain unidentified, leaving the terminal mechanistic steps in EGFR degradation, abscission, and NoCut checkpoint maintenance unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No endosomal or midbody substrate has been identified by cleavage-site mapping or proteomics.","Whether CAPN7 acts catalytically on ESCRT-III subunits themselves or on other midbody factors is unknown.","A full-length CAPN7 structure including the protease domain is lacking."]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,4,5,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[5,6]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[5,6]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4]}],"complexes":[],"partners":["IST1","CHMP1B","HOXA10","AKT1"],"other_free_text":[]},"mechanistic_narrative":"CAPN7 is an atypical calpain family cysteine protease that functions at the interface of endosomal sorting, cytokinetic abscission, and transcription factor turnover. It lacks the classical penta-EF-hand domain but possesses tandem MIT domains that simultaneously engage two distinct MIMs on the ESCRT-III subunit IST1, as revealed by crystal structures and structure-guided mutagenesis; this interaction recruits CAPN7 to EGFR-positive endosomes and to the cytokinetic midbody, where its proteolytic activity is required for efficient EGFR degradation, timely abscission, and maintenance of the NoCut checkpoint [PMID:18316332, PMID:24953135, PMID:36107470, PMID:37772788]. CAPN7 also cleaves PEST-sequence-containing substrates including the transcription factor HOXA10, whose Ca²⁺-dependent degradation by CAPN7 impairs embryo implantation, and AKT1, whose CAPN7-mediated processing enhances AKT1 phosphorylation and promotes FoxO1 nuclear exclusion to negatively regulate endometrial decidualization [PMID:29459744, PMID:35191464]."},"prefetch_data":{"uniprot":{"accession":"Q9Y6W3","full_name":"Calpain-7","aliases":["PalB homolog","PalBH"],"length_aa":813,"mass_kda":92.7,"function":"Calcium-regulated non-lysosomal thiol-protease","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9Y6W3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CAPN7","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/CAPN7","total_profiled":1310},"omim":[{"mim_id":"613530","title":"MUSCULAR DYSTROPHY, LIMB-GIRDLE, TYPE 1H; LGMD1H","url":"https://www.omim.org/entry/613530"},{"mim_id":"606400","title":"CALPAIN 7; CAPN7","url":"https://www.omim.org/entry/606400"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CAPN7"},"hgnc":{"alias_symbol":["PalBH"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y6W3","domains":[{"cath_id":"1.20.58.80","chopping":"4-74","consensus_level":"medium","plddt":82.2928,"start":4,"end":74},{"cath_id":"1.20.58.80","chopping":"83-151","consensus_level":"medium","plddt":95.1046,"start":83,"end":151},{"cath_id":"-","chopping":"215-402","consensus_level":"high","plddt":92.4782,"start":215,"end":402},{"cath_id":"3.90.70.10","chopping":"405-538","consensus_level":"high","plddt":92.1734,"start":405,"end":538},{"cath_id":"2.60.120.380","chopping":"544-682","consensus_level":"high","plddt":92.3482,"start":544,"end":682},{"cath_id":"2.60.120.380","chopping":"689-812","consensus_level":"high","plddt":94.8525,"start":689,"end":812}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6W3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6W3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6W3-F1-predicted_aligned_error_v6.png","plddt_mean":88.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CAPN7","jax_strain_url":"https://www.jax.org/strain/search?query=CAPN7"},"sequence":{"accession":"Q9Y6W3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y6W3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y6W3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6W3"}},"corpus_meta":[{"pmid":"29982135","id":"PMC_29982135","title":"Identification of biomarker candidates for fertility in spermatozoa of crossbred bulls through comparative proteomics.","date":"2018","source":"Theriogenology","url":"https://pubmed.ncbi.nlm.nih.gov/29982135","citation_count":46,"is_preprint":false},{"pmid":"18316332","id":"PMC_18316332","title":"Human calpain 7/PalBH associates with a subset of ESCRT-III-related proteins in its N-terminal region and partly localizes to endocytic membrane compartments.","date":"2008","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18316332","citation_count":27,"is_preprint":false},{"pmid":"36107470","id":"PMC_36107470","title":"Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission.","date":"2022","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/36107470","citation_count":27,"is_preprint":false},{"pmid":"29459744","id":"PMC_29459744","title":"Calpain7 impairs embryo implantation by downregulating β3-integrin expression via degradation of HOXA10.","date":"2018","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/29459744","citation_count":22,"is_preprint":false},{"pmid":"30338799","id":"PMC_30338799","title":"LINC00657 promotes the development of colon cancer by activating PI3K/AKT pathway.","date":"2018","source":"European review for medical and pharmacological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/30338799","citation_count":19,"is_preprint":false},{"pmid":"24953135","id":"PMC_24953135","title":"Involvement of calpain-7 in epidermal growth factor receptor degradation via the endosomal sorting pathway.","date":"2014","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/24953135","citation_count":15,"is_preprint":false},{"pmid":"30910806","id":"PMC_30910806","title":"Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology.","date":"2019","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/30910806","citation_count":12,"is_preprint":false},{"pmid":"35191464","id":"PMC_35191464","title":"Calpain7 negatively regulates human endometrial stromal cell decidualization in EMs by promoting FoxO1 nuclear exclusion via hydrolyzing AKT1.","date":"2022","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/35191464","citation_count":9,"is_preprint":false},{"pmid":"37772788","id":"PMC_37772788","title":"The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission.","date":"2023","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/37772788","citation_count":9,"is_preprint":false},{"pmid":"36685923","id":"PMC_36685923","title":"Selection signature analysis and genome-wide divergence of South African Merino breeds from their founders.","date":"2023","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36685923","citation_count":9,"is_preprint":false},{"pmid":"31814542","id":"PMC_31814542","title":"Structures and functions of penta-EF-hand calcium-binding proteins and their interacting partners: enigmatic relationships between ALG-2 and calpain-7.","date":"2019","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/31814542","citation_count":8,"is_preprint":false},{"pmid":"35565522","id":"PMC_35565522","title":"Identification of Candidate Genes for Pigmentation in Camels Using Genotyping-by-Sequencing.","date":"2022","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/35565522","citation_count":8,"is_preprint":false},{"pmid":"32990242","id":"PMC_32990242","title":"[Mir-29c-3p targeting TUG1 affects migration and invasion of bladder cancer cells by regulating CAPN7 expression].","date":"2020","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/32990242","citation_count":4,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9007,"output_tokens":2396,"usd":0.031481},"stage2":{"model":"claude-opus-4-6","input_tokens":5682,"output_tokens":2290,"usd":0.12849},"total_usd":0.159971,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"CAPN7 (calpain 7/PalBH) interacts with a subset of ESCRT-III-related proteins (CHMPs) through its N-terminal tandem MIT domains; direct interaction with CHMP1B was confirmed by pulldown using recombinant proteins. Endogenous CAPN7 is mostly cytosolic but a fraction localizes to particulate/endosomal compartments, and overexpression of dominant-negative VPS4B causes CAPN7 accumulation in perinuclear puncta that partly co-localize with endocytosed EGF.\",\n      \"method\": \"Pulldown assay (Strep-tagged stable transfectants and recombinant proteins), subcellular fractionation, fluorescence microscopy, GFP-EGF co-localization\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct interaction confirmed with recombinant proteins, multiple orthogonal methods (pulldown, fractionation, live imaging), foundational study\",\n      \"pmids\": [\"18316332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CAPN7 proteolytic activity is required for efficient EGFR degradation via the endosomal sorting pathway. MIT-domain-dependent interaction with ESCRT proteins (including IST1) recruits CAPN7 to EGFR-positive endosomes upon EGF stimulation; a protease-inactive mutant (C290S) acts as dominant-negative and delays EGFR degradation, an effect abolished by deletion of the MIT domains. IST1 knockdown prevents CAPN7(C290S) co-localization with EGFR.\",\n      \"method\": \"CAPN7-knockdown HeLa cells and Capn7-/- MEFs, re-expression of wild-type vs. C290S mutant, MIT domain deletion mutants, immunofluorescence microscopy, EGF-stimulated EGFR degradation assay\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — KO cells + rescue with active-site mutant and domain deletion mutants, multiple orthogonal readouts\",\n      \"pmids\": [\"24953135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CAPN7 degrades HOXA10 in a Ca2+-dependent manner via a PEST sequence in HOXA10; overexpression of CAPN7 in Ishikawa cells or mouse uterus inhibits embryo implantation. Interaction between HOXA10 and CAPN7 represses HOXA10 transcriptional activity and protein stability. Truncation of the PEST motif in HOXA10 abolishes CAPN7-dependent proteolysis; the calpain inhibitor ALLN reverses CAPN7-induced HOXA10 degradation.\",\n      \"method\": \"Overexpression in Ishikawa cells and in vivo mouse uterus model, PEST-motif truncation mutants, calpain inhibitor (ALLN) rescue, in vitro Ca2+-dependent degradation assay, co-immunoprecipitation\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro degradation assay, domain truncation mutants, in vivo rescue with inhibitor, multiple orthogonal methods\",\n      \"pmids\": [\"29459744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CAPN7 mediates protease-dependent degradation of SMN (survival motor neuron) protein; the cysteine protease inhibitor Z-FA-FMK stabilizes SMN by blocking CAPN7 (among CAPN1, CTSB, and CTSL) activity, identifying CAPN7 as one of the proteases responsible for SMN turnover.\",\n      \"method\": \"SMN2-GFP reporter drug screen, cysteine protease inhibitor treatment, Western blot analysis of SMN levels, identification of CAPN7 as mediator of SMN degradation\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pharmacological inhibitor approach without direct substrate confirmation or mutagenesis specific to CAPN7; CAPN7 identified as one of four proteases\",\n      \"pmids\": [\"30910806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CAPN7 hydrolyzes AKT1 (which contains a conserved PEST sequence) and enhances AKT1 phosphorylation, thereby promoting phosphorylation of the downstream transcription factor FoxO1 at Ser319 and increasing FoxO1 nuclear exclusion, which attenuates FoxO1 transcriptional activity and negatively regulates decidualization of human endometrial stromal cells.\",\n      \"method\": \"CAPN7 overexpression/knockdown in HESCs, in vitro and in vivo decidualization models, PEST sequence identification in AKT1, co-immunoprecipitation, phospho-specific Western blotting, nuclear/cytoplasmic fractionation\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — PEST-dependent cleavage supported by IP and phospho-blots, in vivo phenotype, but reconstituted in vitro cleavage not fully reported\",\n      \"pmids\": [\"35191464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CAPN7 localizes to cytokinetic midbody membrane bridges through interaction with its specific ESCRT-III binding partner IST1, and is required for both efficient cytokinetic abscission and maintenance of the NoCut abscission checkpoint. Quantitative mapping of MIT–ESCRT-III interactions showed the CAPN7 MIT domain binds IST1 among 228 pairwise interactions tested.\",\n      \"method\": \"Comprehensive quantitative MIT–ESCRT-III interaction mapping (228 pairwise), live-cell imaging of cytokinetic midbodies, CAPN7 depletion/rescue abscission assays, NoCut checkpoint assays, crystal structure of SPASTIN-IST1 (as part of same study)\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — comprehensive quantitative interactome, functional abscission assays, checkpoint assays, replicated across multiple MIT enzymes in same study\",\n      \"pmids\": [\"36107470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The tandem MIT domains of CAPN7 bind simultaneously to two distinct MIT-interaction motifs (MIMs) of IST1; crystal structures of the CAPN7 tandem-MIT–IST1 complex were solved, and structure-guided point mutations in either MIT domain disrupted IST1 binding in vitro and in cells. Depletion/rescue experiments demonstrated that the CAPN7–IST1 interaction is required for (1) CAPN7 recruitment to midbodies, (2) efficient abscission, and (3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also independently required for abscission and checkpoint maintenance.\",\n      \"method\": \"Crystallography (tandem MIT–IST1 complex), structure-guided mutagenesis, in vitro binding assays, CAPN7 depletion/rescue in cells, abscission timing assays, NoCut checkpoint assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis plus functional cellular rescue with multiple orthogonal readouts\",\n      \"pmids\": [\"37772788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CAPN7 lacks a penta-EF-hand domain but contains two tandem MIT domains that interact with a subset of ESCRT-III proteins to regulate EGF receptor downregulation, distinguishing its mechanism from classical calpains.\",\n      \"method\": \"Review/synthesis of structural and functional data including MIT domain interaction studies and EGFR degradation experiments\",\n      \"journal\": \"Bioscience, biotechnology, and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — review article synthesizing prior experimental work; no new primary experiments\",\n      \"pmids\": [\"31814542\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CAPN7 is an atypical cysteine protease that lacks a penta-EF-hand domain but contains tandem MIT domains, which directly bind ESCRT-III subunits (principally IST1, via two distinct MIMs simultaneously) to recruit CAPN7 to endosomes and the cytokinetic midbody; there, its proteolytic activity is required for EGFR degradation via the endosomal sorting pathway and for both efficient cytokinetic abscission and NoCut checkpoint maintenance, and it can also cleave substrates containing PEST sequences (including HOXA10 and AKT1) to modulate transcription factor stability and signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CAPN7 is an atypical calpain family cysteine protease that functions at the interface of endosomal sorting, cytokinetic abscission, and transcription factor turnover. It lacks the classical penta-EF-hand domain but possesses tandem MIT domains that simultaneously engage two distinct MIMs on the ESCRT-III subunit IST1, as revealed by crystal structures and structure-guided mutagenesis; this interaction recruits CAPN7 to EGFR-positive endosomes and to the cytokinetic midbody, where its proteolytic activity is required for efficient EGFR degradation, timely abscission, and maintenance of the NoCut checkpoint [PMID:18316332, PMID:24953135, PMID:36107470, PMID:37772788]. CAPN7 also cleaves PEST-sequence-containing substrates including the transcription factor HOXA10, whose Ca²⁺-dependent degradation by CAPN7 impairs embryo implantation, and AKT1, whose CAPN7-mediated processing enhances AKT1 phosphorylation and promotes FoxO1 nuclear exclusion to negatively regulate endometrial decidualization [PMID:29459744, PMID:35191464].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Establishing that CAPN7 physically engages the ESCRT-III machinery resolved how an atypical calpain without a penta-EF-hand domain could be targeted to endosomal membranes.\",\n      \"evidence\": \"Recombinant pulldown confirmed direct CAPN7–CHMP1B interaction via tandem MIT domains; subcellular fractionation and fluorescence microscopy showed partial endosomal localization enhanced by dominant-negative VPS4B in HeLa cells.\",\n      \"pmids\": [\"18316332\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Proteolytic activity was not tested; whether ESCRT-III binding serves a catalytic or scaffolding purpose remained open.\",\n        \"The preferred ESCRT-III partner(s) beyond CHMP1B were not ranked quantitatively.\",\n        \"Endogenous substrate(s) at endosomes were unknown.\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating that CAPN7 protease activity is required for EGFR degradation linked the MIT-domain-mediated ESCRT-III recruitment to a specific endosomal sorting function.\",\n      \"evidence\": \"CAPN7 knockdown and Capn7−/− MEFs showed delayed EGF-stimulated EGFR degradation; rescue with catalytic-dead C290S mutant acted dominant-negatively, an effect abolished by MIT domain deletion; IST1 knockdown prevented CAPN7(C290S) co-localization with EGFR in HeLa cells.\",\n      \"pmids\": [\"24953135\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The direct endosomal substrate(s) cleaved by CAPN7 to facilitate EGFR degradation were not identified.\",\n        \"Whether CAPN7 acts on EGFR itself or on ESCRT-associated factors was unresolved.\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identifying HOXA10 as a direct PEST-dependent substrate expanded CAPN7 function beyond endosomal sorting to transcription factor turnover and reproductive biology.\",\n      \"evidence\": \"In vitro Ca²⁺-dependent degradation assay, PEST-motif truncation abolished cleavage, calpain inhibitor ALLN rescued HOXA10 stability; CAPN7 overexpression in mouse uterus inhibited embryo implantation.\",\n      \"pmids\": [\"29459744\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The precise cleavage site(s) within HOXA10 were not mapped.\",\n        \"Whether CAPN7-mediated HOXA10 degradation operates via endosomal or cytosolic pools was unclear.\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Systematic MIT–ESCRT-III interaction mapping identified IST1 as the principal CAPN7 binding partner and showed that this interaction recruits CAPN7 to the cytokinetic midbody for abscission and NoCut checkpoint control, revealing a second major cellular function beyond endosomal sorting.\",\n      \"evidence\": \"Quantitative pairwise mapping of 228 MIT–ESCRT-III interactions, live-cell abscission timing assays, and NoCut checkpoint assays upon CAPN7 depletion/rescue.\",\n      \"pmids\": [\"36107470\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The midbody substrate(s) of CAPN7 protease activity were not identified.\",\n        \"Whether the abscission and checkpoint functions require the same or different proteolytic events was unresolved.\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrating that CAPN7 cleaves the PEST-containing kinase AKT1 and modulates FoxO1 phosphorylation extended the substrate repertoire to a major signaling node controlling endometrial decidualization.\",\n      \"evidence\": \"CAPN7 overexpression/knockdown in human endometrial stromal cells, co-immunoprecipitation with AKT1, phospho-specific Western blotting for FoxO1, nuclear/cytoplasmic fractionation.\",\n      \"pmids\": [\"35191464\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Reconstituted in vitro cleavage of AKT1 by purified CAPN7 was not fully demonstrated.\",\n        \"The precise AKT1 cleavage site and whether this generates a constitutively active fragment were not determined.\",\n        \"The relationship between AKT1 hydrolysis and enhanced AKT1 phosphorylation is mechanistically unclear.\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Crystal structures of the CAPN7 tandem-MIT–IST1 complex revealed a bipartite binding mode in which both MIT domains simultaneously engage two MIMs on IST1, providing the structural basis for CAPN7 recruitment to midbodies and its roles in abscission and the NoCut checkpoint.\",\n      \"evidence\": \"X-ray crystallography of tandem-MIT–IST1 complex, structure-guided point mutations disrupting IST1 binding in vitro and in cells, depletion/rescue abscission and checkpoint assays demonstrating that both MIT-mediated recruitment and catalytic activity are independently required.\",\n      \"pmids\": [\"37772788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The midbody substrate(s) whose cleavage mediates abscission timing and checkpoint signaling remain unidentified.\",\n        \"How catalytic activity and IST1-mediated recruitment are coordinated is not structurally resolved.\",\n        \"Whether additional ESCRT-III subunits contribute to CAPN7 regulation at the midbody in vivo is unknown.\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct proteolytic substrates of CAPN7 at endosomes and at the cytokinetic midbody remain unidentified, leaving the terminal mechanistic steps in EGFR degradation, abscission, and NoCut checkpoint maintenance unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No endosomal or midbody substrate has been identified by cleavage-site mapping or proteomics.\",\n        \"Whether CAPN7 acts catalytically on ESCRT-III subunits themselves or on other midbody factors is unknown.\",\n        \"A full-length CAPN7 structure including the protease domain is lacking.\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 4, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"IST1\",\n      \"CHMP1B\",\n      \"HOXA10\",\n      \"AKT1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}