{"doi":"10.1016/j.jbc.2025.110243","title":"Quantification and structure–function analysis of calpain-1 and calpain-2 protease subunit interactions","abstract":"Calpain-1 and calpain-2 are heterodimeric proteases consisting of a common small regulatory subunit CAPNS1 and a large catalytic subunit, CAPN1 or CAPN2, respectively. These calpains have emerged as potential therapeutic targets in cancer and other diseases through their roles in cell signaling pathways affecting sensitivity to chemotherapeutic and targeted drugs, and in promoting metastasis. While inhibition of calpains has the potential to provide therapeutic benefit to cancer patients, there are currently no clinically approved active site directed drugs that specifically and effectively inhibit them. However, the structures of calpain-1 and calpain-2 make them susceptible to allosteric inhibition aimed at interfering with heterodimerization of the catalytic and regulatory subunits, which is necessary for stability and proteolytic activity. Split-Nanoluciferase biosensors were generated to quantify the protein-protein interactions (PPIs) between the calcium-binding penta-EF hand (PEF) domains of CAPN1 or CAPN2 and CAPNS1. These biosensors were used to quantify the heterodimer dissociation constants (K D ) of calpain-1 and calpain-2, estimated at 185 nM and 509 nM, respectively, in the presence of 5 mM Ca 2+ ; and 362 nM and 1651 nM, respectively, in the presence of Mg 2+ . The half-maximal Ca 2+ concentrations supporting these PPIs for calpain-1 and calpain-2 were 59.9 μM and 940.8 μM, respectively. Molecular modeling, based on the crystal structure of calpain-2, was used to predict 20 residues of the PEF domains that contribute to heterodimerization. Individual point mutation of CAPNS1 at Q263 reduced the catalytic activity of calpain-2 to 51.0 ± 6.4 % in live cells.","journal":"Journal of Biological Chemistry","year":2025,"id":514731,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":7,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9582,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1069305,"name":"Prawin Rimal","orcid":null,"position":1,"is_corresponding":false},{"id":901843,"name":"Pitambar Poudel","orcid":"0000-0001-8894-3342","position":2,"is_corresponding":false},{"id":1378786,"name":"Victoria Lewtas","orcid":null,"position":3,"is_corresponding":false},{"id":1378787,"name":"Mathias Bell","orcid":null,"position":4,"is_corresponding":false},{"id":449225,"name":"Shailesh Kumar Panday","orcid":"0000-0003-3099-8679","position":5,"is_corresponding":false},{"id":1378087,"name":"Brian J. Laight","orcid":"0000-0002-0371-3335","position":6,"is_corresponding":false},{"id":1378088,"name":"Danielle Harper","orcid":"0009-0009-1184-4923","position":7,"is_corresponding":false},{"id":1378089,"name":"Stacy Grieve","orcid":"0000-0003-3090-5583","position":8,"is_corresponding":false},{"id":899846,"name":"George S. Baillie","orcid":"0000-0003-2469-6316","position":9,"is_corresponding":false},{"id":1378090,"name":"Kazem Nouri","orcid":"0000-0001-7787-1304","position":10,"is_corresponding":false},{"id":1074625,"name":"Peter L. Davies","orcid":"0000-0002-8026-7818","position":11,"is_corresponding":false},{"id":268022,"name":"Emil Alexov","orcid":"0000-0001-5346-0156","position":12,"is_corresponding":false},{"id":15840,"name":"Peter A. Greer","orcid":"0000-0002-7809-5105","position":13,"is_corresponding":false},{"id":1378785,"name":"Іван Шаповалов","orcid":null,"position":0,"is_corresponding":true}],"reference_count":65,"raw_metadata":null,"created_at":"2026-07-19T02:48:34.431522Z","pmid":"40383147","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}