{"gene":"CAPN7","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2008,"finding":"CAPN7 interacts with a subset of ESCRT-III/CHMP proteins (including CHMP1B) through its N-terminal tandem MIT domains; this interaction is necessary and sufficient for CHMP binding. Most endogenous CAPN7 is cytosolic, but a small fraction associates with particulate (membrane) fractions and co-localizes with endocytosed EGF, implicating it in the endosomal pathway.","method":"Pulldown assays using Strep-tagged stable HEK293T transfectants, recombinant protein pulldown for direct interaction confirmation, fluorescence microscopy with dominant-negative SKD1/Vps4B, subcellular fractionation, co-localization with endocytosed TMR-EGF","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal pulldowns with recombinant proteins confirming direct interaction, domain mapping, subcellular fractionation, and live imaging all in one study; replicated conceptually by later papers","pmids":["18316332"],"is_preprint":false},{"year":2014,"finding":"CAPN7 proteolytic activity accelerates EGFR degradation via the endosomal sorting pathway. The MIT domains recruit CAPN7 to EGFR-positive endosomes via IST1; IST1 knockdown abolishes CAPN7 co-localization with EGFR. A protease-inactive mutant (C290S) acts as dominant-negative, while MIT-domain deletion abolishes this dominant-negative effect, demonstrating that both protease activity and MIT-domain/ESCRT interaction are required.","method":"CAPN7 knockdown in HeLa cells and Capn7−/− MEFs with EGFR degradation assay, re-expression of wild-type vs. C290S mutant, immunofluorescence microscopy, IST1 knockdown epistasis","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function KO/KD with defined molecular phenotype, rescue with WT vs. catalytic mutant, domain-deletion mutant analysis, and epistasis with IST1; multiple orthogonal methods in one study","pmids":["24953135"],"is_preprint":false},{"year":2018,"finding":"CAPN7 degrades HOXA10 in a Ca2+-dependent manner via a PEST sequence in HOXA10, reducing HOXA10 protein stability and transcriptional activity, which in turn downregulates β3-integrin expression and impairs embryo implantation. The calpain inhibitor ALLN reverses this degradation, and truncation of the PEST motif in HOXA10 abolishes CAPN7-dependent proteolysis.","method":"CAPN7 overexpression in Ishikawa cells and mouse uterus, in vitro embryo implantation assay, in vivo implantation assay, co-immunoprecipitation, ALLN inhibitor rescue, HOXA10 PEST motif truncation mutant, luciferase transcriptional activity assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo loss/gain-of-function with defined substrate (HOXA10 PEST motif), inhibitor rescue, and truncation mutagenesis; single lab","pmids":["29459744"],"is_preprint":false},{"year":2019,"finding":"CAPN7 (along with CAPN1, CTSB, CTSL) mediates proteolytic degradation of SMN protein (both full-length and exon-7-deleted forms); inhibition by the cysteine protease inhibitor Z-FA-FMK stabilizes SMN.","method":"SMN2-GFP reporter drug screen, cysteine protease inhibitor treatment, Western blot for SMN levels, knockdown of individual proteases","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — SMN degradation attributed to CAPN7 by knockdown and inhibitor experiments in a reporter cell line; functional phenotype demonstrated but single lab and limited mechanistic dissection of CAPN7 specifically","pmids":["30910806"],"is_preprint":false},{"year":2022,"finding":"CAPN7 localizes to the cytokinetic midbody membrane bridge through its MIT domain interaction specifically with IST1 (not other ESCRT-III subunits), and is required for efficient cytokinetic abscission and for maintenance of the NoCut abscission checkpoint.","method":"Comprehensive quantitative MIT-domain/ESCRT-III tail interaction mapping (228 pairwise interactions), crystal structure of SPASTIN MIT–IST1 tail complex, live-cell localization of CAPN7 to midbody, abscission timing assay, NoCut checkpoint assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — quantitative interactome mapping, crystal structure of related MIT-ESCRT-III complex, functional abscission and checkpoint assays; two independent labs (Sundquist/Ullman groups)","pmids":["36107470"],"is_preprint":false},{"year":2022,"finding":"CAPN7 hydrolyzes AKT1 (which contains a PEST sequence) and enhances AKT1 phosphorylation, leading to increased phosphorylation of FoxO1 at Ser319, nuclear exclusion of FoxO1, and attenuation of FoxO1 transcriptional activity, thereby negatively regulating human endometrial stromal cell decidualization.","method":"CAPN7 knockdown and overexpression in HESCs, in vitro decidualization assay, in vivo mouse model, co-immunoprecipitation, phospho-specific Western blotting for AKT1 and FoxO1, nuclear/cytoplasmic fractionation, FoxO1 transcriptional reporter","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain/loss-of-function with defined substrate (AKT1 PEST sequence), downstream phospho-signaling readout, and in vivo validation; single lab","pmids":["35191464"],"is_preprint":false},{"year":2023,"finding":"The tandem MIT domains of CAPN7 bind simultaneously to two distinct MIT-interaction motifs (MIM1 and MIM2) within IST1; structure-guided point mutations in either MIT domain disrupt IST1 binding in vitro and in cells. 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 of CAPN7 tandem MIT domains bound to IST1 MIM peptides, ITC/biochemical binding assays, structure-guided mutagenesis, depletion/rescue experiments in cells, abscission timing assay, NoCut checkpoint assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by mutagenesis, in vitro binding assays, and cell-based rescue experiments; multiple orthogonal methods in one rigorous study","pmids":["37772788"],"is_preprint":false}],"current_model":"CAPN7 is a cysteine protease that uses its tandem N-terminal MIT domains to bind the ESCRT-III subunit IST1 at two distinct interaction motifs, which recruits CAPN7 to endosomal membranes and cytokinetic midbodies; at endosomes its proteolytic activity accelerates EGFR degradation via the ESCRT pathway, and at the midbody it is required for both efficient cytokinetic abscission and NoCut checkpoint maintenance, while in non-dividing contexts its protease activity also degrades substrates such as HOXA10 and AKT1 via their PEST sequences to modulate transcriptional and signaling outputs."},"narrative":{"mechanistic_narrative":"CAPN7 is a cysteine protease that functions at the interface of the ESCRT membrane-remodeling machinery and substrate-specific proteolysis [PMID:18316332, PMID:24953135, PMID:36107470]. Its tandem N-terminal MIT domains bind ESCRT-III/CHMP subunits, an interaction that is both necessary and sufficient for CHMP binding and that targets a membrane-associated pool of CAPN7 to the endosomal pathway, where it co-localizes with endocytosed EGF [PMID:18316332]. The MIT domains engage the ESCRT-III subunit IST1 specifically, with the two domains binding simultaneously to distinct MIM1 and MIM2 motifs within IST1; this interaction recruits CAPN7 to EGFR-positive endosomes and to the cytokinetic midbody [PMID:24953135, PMID:36107470, PMID:37772788]. At endosomes, CAPN7 proteolytic activity accelerates EGFR degradation, with both the catalytic cysteine (C290) and MIT/IST1 engagement required [PMID:24953135]. At the midbody, both IST1 binding and protease activity are independently required for efficient abscission and for maintenance of the NoCut abscission checkpoint [PMID:36107470, PMID:37772788]. In other contexts CAPN7 cleaves substrates bearing PEST sequences: it degrades HOXA10 in a Ca2+-dependent, inhibitor-sensitive manner to reduce β3-integrin expression and impair embryo implantation [PMID:29459744], and it acts on AKT1 to enhance AKT1 signaling, drive FoxO1 phosphorylation and nuclear exclusion, and restrain endometrial stromal cell decidualization [PMID:35191464]. CAPN7 also contributes to proteolytic turnover of SMN protein [PMID:30910806].","teleology":[{"year":2008,"claim":"Established that CAPN7 physically couples to the ESCRT-III machinery and accesses the endosomal pathway, identifying the structural basis for its membrane recruitment.","evidence":"Recombinant and Strep-tagged protein pulldowns mapping the interaction to the tandem MIT domains, subcellular fractionation, and co-localization with endocytosed EGF in HEK293T/microscopy","pmids":["18316332"],"confidence":"High","gaps":["Did not demonstrate a proteolytic function at endosomes","Did not identify which specific CHMP/ESCRT-III subunit drives physiological recruitment"]},{"year":2014,"claim":"Showed that CAPN7 is not merely an ESCRT passenger but a functional protease in endosomal sorting, accelerating EGFR degradation through combined catalytic and adaptor activities.","evidence":"Knockdown in HeLa and Capn7−/− MEFs with EGFR degradation assays, rescue with wild-type vs C290S catalytic mutant, MIT-deletion analysis, and IST1 knockdown epistasis","pmids":["24953135"],"confidence":"High","gaps":["Did not identify the direct proteolytic substrate at the endosome","Mechanism by which proteolysis accelerates sorting not resolved"]},{"year":2018,"claim":"Identified the first defined substrate motif, demonstrating CAPN7 degrades PEST-containing HOXA10 to control a transcriptional program governing embryo implantation.","evidence":"CAPN7 overexpression in Ishikawa cells and mouse uterus, co-IP, ALLN inhibitor rescue, HOXA10 PEST truncation, luciferase and implantation assays","pmids":["29459744"],"confidence":"Medium","gaps":["Single lab","Ca2+-dependence of CAPN7 catalysis not biochemically reconstituted","Direct cleavage site not mapped"]},{"year":2019,"claim":"Implicated CAPN7 among redundant cysteine proteases that turn over SMN protein, expanding its substrate range.","evidence":"SMN2-GFP reporter drug screen, cysteine protease inhibitor treatment, and knockdown of individual proteases with SMN Western blot","pmids":["30910806"],"confidence":"Medium","gaps":["CAPN7-specific contribution confounded by parallel proteases (CAPN1, CTSB, CTSL)","No direct biochemical demonstration of CAPN7 cleaving SMN","No cleavage site or motif defined"]},{"year":2022,"claim":"Extended CAPN7's ESCRT role to cell division, placing it at the midbody where IST1-specific recruitment supports abscission and the NoCut checkpoint.","evidence":"Quantitative MIT-domain/ESCRT-III interaction mapping, related MIT-IST1 crystal structure, midbody live imaging, abscission timing, and NoCut checkpoint assays (Sundquist/Ullman groups)","pmids":["36107470"],"confidence":"High","gaps":["Did not resolve whether protease activity acts on a midbody substrate","Identity of any cleaved checkpoint regulator unknown"]},{"year":2022,"claim":"Defined a second PEST substrate, AKT1, linking CAPN7 proteolysis to phospho-signaling output and FoxO1-dependent transcription in decidualization.","evidence":"CAPN7 knockdown/overexpression in HESCs, co-IP, phospho-Western for AKT1/FoxO1, nuclear-cytoplasmic fractionation, FoxO1 reporter, and in vivo mouse model","pmids":["35191464"],"confidence":"Medium","gaps":["Single lab","How hydrolysis of AKT1 enhances its phosphorylation mechanistically unclear","Direct cleavage site not mapped"]},{"year":2023,"claim":"Provided the atomic basis for CAPN7 recruitment, showing the two MIT domains clamp distinct MIM1/MIM2 motifs on IST1 and dissecting IST1-binding from catalytic requirements at the midbody.","evidence":"Crystallography of CAPN7 tandem MIT domains bound to IST1 MIM peptides, ITC binding, structure-guided mutagenesis, and depletion/rescue abscission and NoCut checkpoint assays","pmids":["37772788"],"confidence":"High","gaps":["Did not identify the midbody proteolytic substrate","Did not resolve the full-length CAPN7 structure or catalytic-domain regulation"]},{"year":null,"claim":"The direct proteolytic substrates of CAPN7 at endosomes and midbodies, and the regulation of its catalytic activity, remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No endosomal or midbody cleavage substrate identified","Ca2+- and context-dependent regulation of catalysis not reconstituted","No full-length structure linking MIT recruitment to protease domain"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,5]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[4,6]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,5]}],"complexes":[],"partners":["IST1","CHMP1B","HOXA10","AKT1"],"other_free_text":[]}},"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":"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":28,"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":"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":13,"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":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":"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":"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":"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":"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":9765,"output_tokens":2226,"usd":0.031343,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9281,"output_tokens":2961,"usd":0.060215,"stage2_stop_reason":"end_turn"},"total_usd":0.091558,"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\": 2008,\n      \"finding\": \"CAPN7 interacts with a subset of ESCRT-III/CHMP proteins (including CHMP1B) through its N-terminal tandem MIT domains; this interaction is necessary and sufficient for CHMP binding. Most endogenous CAPN7 is cytosolic, but a small fraction associates with particulate (membrane) fractions and co-localizes with endocytosed EGF, implicating it in the endosomal pathway.\",\n      \"method\": \"Pulldown assays using Strep-tagged stable HEK293T transfectants, recombinant protein pulldown for direct interaction confirmation, fluorescence microscopy with dominant-negative SKD1/Vps4B, subcellular fractionation, co-localization with endocytosed TMR-EGF\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal pulldowns with recombinant proteins confirming direct interaction, domain mapping, subcellular fractionation, and live imaging all in one study; replicated conceptually by later papers\",\n      \"pmids\": [\"18316332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CAPN7 proteolytic activity accelerates EGFR degradation via the endosomal sorting pathway. The MIT domains recruit CAPN7 to EGFR-positive endosomes via IST1; IST1 knockdown abolishes CAPN7 co-localization with EGFR. A protease-inactive mutant (C290S) acts as dominant-negative, while MIT-domain deletion abolishes this dominant-negative effect, demonstrating that both protease activity and MIT-domain/ESCRT interaction are required.\",\n      \"method\": \"CAPN7 knockdown in HeLa cells and Capn7−/− MEFs with EGFR degradation assay, re-expression of wild-type vs. C290S mutant, immunofluorescence microscopy, IST1 knockdown epistasis\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function KO/KD with defined molecular phenotype, rescue with WT vs. catalytic mutant, domain-deletion mutant analysis, and epistasis with IST1; multiple orthogonal methods in one study\",\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, reducing HOXA10 protein stability and transcriptional activity, which in turn downregulates β3-integrin expression and impairs embryo implantation. The calpain inhibitor ALLN reverses this degradation, and truncation of the PEST motif in HOXA10 abolishes CAPN7-dependent proteolysis.\",\n      \"method\": \"CAPN7 overexpression in Ishikawa cells and mouse uterus, in vitro embryo implantation assay, in vivo implantation assay, co-immunoprecipitation, ALLN inhibitor rescue, HOXA10 PEST motif truncation mutant, luciferase transcriptional activity assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo loss/gain-of-function with defined substrate (HOXA10 PEST motif), inhibitor rescue, and truncation mutagenesis; single lab\",\n      \"pmids\": [\"29459744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CAPN7 (along with CAPN1, CTSB, CTSL) mediates proteolytic degradation of SMN protein (both full-length and exon-7-deleted forms); inhibition by the cysteine protease inhibitor Z-FA-FMK stabilizes SMN.\",\n      \"method\": \"SMN2-GFP reporter drug screen, cysteine protease inhibitor treatment, Western blot for SMN levels, knockdown of individual proteases\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — SMN degradation attributed to CAPN7 by knockdown and inhibitor experiments in a reporter cell line; functional phenotype demonstrated but single lab and limited mechanistic dissection of CAPN7 specifically\",\n      \"pmids\": [\"30910806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CAPN7 localizes to the cytokinetic midbody membrane bridge through its MIT domain interaction specifically with IST1 (not other ESCRT-III subunits), and is required for efficient cytokinetic abscission and for maintenance of the NoCut abscission checkpoint.\",\n      \"method\": \"Comprehensive quantitative MIT-domain/ESCRT-III tail interaction mapping (228 pairwise interactions), crystal structure of SPASTIN MIT–IST1 tail complex, live-cell localization of CAPN7 to midbody, abscission timing assay, NoCut checkpoint assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — quantitative interactome mapping, crystal structure of related MIT-ESCRT-III complex, functional abscission and checkpoint assays; two independent labs (Sundquist/Ullman groups)\",\n      \"pmids\": [\"36107470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CAPN7 hydrolyzes AKT1 (which contains a PEST sequence) and enhances AKT1 phosphorylation, leading to increased phosphorylation of FoxO1 at Ser319, nuclear exclusion of FoxO1, and attenuation of FoxO1 transcriptional activity, thereby negatively regulating human endometrial stromal cell decidualization.\",\n      \"method\": \"CAPN7 knockdown and overexpression in HESCs, in vitro decidualization assay, in vivo mouse model, co-immunoprecipitation, phospho-specific Western blotting for AKT1 and FoxO1, nuclear/cytoplasmic fractionation, FoxO1 transcriptional reporter\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain/loss-of-function with defined substrate (AKT1 PEST sequence), downstream phospho-signaling readout, and in vivo validation; single lab\",\n      \"pmids\": [\"35191464\"],\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 (MIM1 and MIM2) within IST1; structure-guided point mutations in either MIT domain disrupt IST1 binding in vitro and in cells. 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 of CAPN7 tandem MIT domains bound to IST1 MIM peptides, ITC/biochemical binding assays, structure-guided mutagenesis, depletion/rescue experiments in cells, abscission timing assay, NoCut checkpoint assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by mutagenesis, in vitro binding assays, and cell-based rescue experiments; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"37772788\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CAPN7 is a cysteine protease that uses its tandem N-terminal MIT domains to bind the ESCRT-III subunit IST1 at two distinct interaction motifs, which recruits CAPN7 to endosomal membranes and cytokinetic midbodies; at endosomes its proteolytic activity accelerates EGFR degradation via the ESCRT pathway, and at the midbody it is required for both efficient cytokinetic abscission and NoCut checkpoint maintenance, while in non-dividing contexts its protease activity also degrades substrates such as HOXA10 and AKT1 via their PEST sequences to modulate transcriptional and signaling outputs.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CAPN7 is a cysteine protease that functions at the interface of the ESCRT membrane-remodeling machinery and substrate-specific proteolysis [#0, #1, #4]. Its tandem N-terminal MIT domains bind ESCRT-III/CHMP subunits, an interaction that is both necessary and sufficient for CHMP binding and that targets a membrane-associated pool of CAPN7 to the endosomal pathway, where it co-localizes with endocytosed EGF [#0]. The MIT domains engage the ESCRT-III subunit IST1 specifically, with the two domains binding simultaneously to distinct MIM1 and MIM2 motifs within IST1; this interaction recruits CAPN7 to EGFR-positive endosomes and to the cytokinetic midbody [#1, #4, #6]. At endosomes, CAPN7 proteolytic activity accelerates EGFR degradation, with both the catalytic cysteine (C290) and MIT/IST1 engagement required [#1]. At the midbody, both IST1 binding and protease activity are independently required for efficient abscission and for maintenance of the NoCut abscission checkpoint [#4, #6]. In other contexts CAPN7 cleaves substrates bearing PEST sequences: it degrades HOXA10 in a Ca2+-dependent, inhibitor-sensitive manner to reduce \\u03b23-integrin expression and impair embryo implantation [#2], and it acts on AKT1 to enhance AKT1 signaling, drive FoxO1 phosphorylation and nuclear exclusion, and restrain endometrial stromal cell decidualization [#5]. CAPN7 also contributes to proteolytic turnover of SMN protein [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established that CAPN7 physically couples to the ESCRT-III machinery and accesses the endosomal pathway, identifying the structural basis for its membrane recruitment.\",\n      \"evidence\": \"Recombinant and Strep-tagged protein pulldowns mapping the interaction to the tandem MIT domains, subcellular fractionation, and co-localization with endocytosed EGF in HEK293T/microscopy\",\n      \"pmids\": [\"18316332\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not demonstrate a proteolytic function at endosomes\", \"Did not identify which specific CHMP/ESCRT-III subunit drives physiological recruitment\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed that CAPN7 is not merely an ESCRT passenger but a functional protease in endosomal sorting, accelerating EGFR degradation through combined catalytic and adaptor activities.\",\n      \"evidence\": \"Knockdown in HeLa and Capn7\\u2212/\\u2212 MEFs with EGFR degradation assays, rescue with wild-type vs C290S catalytic mutant, MIT-deletion analysis, and IST1 knockdown epistasis\",\n      \"pmids\": [\"24953135\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the direct proteolytic substrate at the endosome\", \"Mechanism by which proteolysis accelerates sorting not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified the first defined substrate motif, demonstrating CAPN7 degrades PEST-containing HOXA10 to control a transcriptional program governing embryo implantation.\",\n      \"evidence\": \"CAPN7 overexpression in Ishikawa cells and mouse uterus, co-IP, ALLN inhibitor rescue, HOXA10 PEST truncation, luciferase and implantation assays\",\n      \"pmids\": [\"29459744\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Ca2+-dependence of CAPN7 catalysis not biochemically reconstituted\", \"Direct cleavage site not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Implicated CAPN7 among redundant cysteine proteases that turn over SMN protein, expanding its substrate range.\",\n      \"evidence\": \"SMN2-GFP reporter drug screen, cysteine protease inhibitor treatment, and knockdown of individual proteases with SMN Western blot\",\n      \"pmids\": [\"30910806\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CAPN7-specific contribution confounded by parallel proteases (CAPN1, CTSB, CTSL)\", \"No direct biochemical demonstration of CAPN7 cleaving SMN\", \"No cleavage site or motif defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended CAPN7's ESCRT role to cell division, placing it at the midbody where IST1-specific recruitment supports abscission and the NoCut checkpoint.\",\n      \"evidence\": \"Quantitative MIT-domain/ESCRT-III interaction mapping, related MIT-IST1 crystal structure, midbody live imaging, abscission timing, and NoCut checkpoint assays (Sundquist/Ullman groups)\",\n      \"pmids\": [\"36107470\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether protease activity acts on a midbody substrate\", \"Identity of any cleaved checkpoint regulator unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined a second PEST substrate, AKT1, linking CAPN7 proteolysis to phospho-signaling output and FoxO1-dependent transcription in decidualization.\",\n      \"evidence\": \"CAPN7 knockdown/overexpression in HESCs, co-IP, phospho-Western for AKT1/FoxO1, nuclear-cytoplasmic fractionation, FoxO1 reporter, and in vivo mouse model\",\n      \"pmids\": [\"35191464\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"How hydrolysis of AKT1 enhances its phosphorylation mechanistically unclear\", \"Direct cleavage site not mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided the atomic basis for CAPN7 recruitment, showing the two MIT domains clamp distinct MIM1/MIM2 motifs on IST1 and dissecting IST1-binding from catalytic requirements at the midbody.\",\n      \"evidence\": \"Crystallography of CAPN7 tandem MIT domains bound to IST1 MIM peptides, ITC binding, structure-guided mutagenesis, and depletion/rescue abscission and NoCut checkpoint assays\",\n      \"pmids\": [\"37772788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the midbody proteolytic substrate\", \"Did not resolve the full-length CAPN7 structure or catalytic-domain regulation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct proteolytic substrates of CAPN7 at endosomes and midbodies, and the regulation of its catalytic activity, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No endosomal or midbody cleavage substrate identified\", \"Ca2+- and context-dependent regulation of catalysis not reconstituted\", \"No full-length structure linking MIT recruitment to protease domain\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 5]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"IST1\", \"CHMP1B\", \"HOXA10\", \"AKT1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}