The Identification of Proteolytic Substrates of Calpain-5 with N-Terminomics.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:40650235", "kind": "science-record", "revision": 2, "content_hash": "sha256:05a17bdc2e5c58195cc8164d0b4206489a26ccc1b12276a72bba72912ee3b768", "created_at": "2026-11-01T20:30:44Z", "updated_at": "2026-11-01T21:35:26Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "The Identification of Proteolytic Substrates of Calpain-5 with N-Terminomics.", "doi": "10.3390/ijms26136459", "pmid": "40650235", "authors": ["Jozsef Gal", "Antoine Dufour", "Daniel Young", "Eddy S Yang", "James W Geddes"], "journal": "International journal of molecular sciences", "year": 2025, "abstract": "Calpain-5/CAPN5 is a calcium-activated, non-lysosomal cysteine (thiol) protease. The substrate repertoire of CAPN5 is not known. Calpains catalyze limited proteolysis of their substrates, generating neo-N-termini that correspond to internal residues of their nascent substrate proteins. To identify such neo-N-termini generated by CAPN5, we employed an N-terminomics approach called TAILS (Terminal amine isotopic labeling of substrates) to quantitatively compare the N-terminal peptides detected in parental and CAPN5-deficient SH-SY5Y neuroblastoma cells. Thirty neo-N-termini corresponding to 29 protein groups and 24 unique proteins were detected to be depleted in the CAPN5-/- cells. A subset of the identified putative substrates was further studied with CAPN5 co-immunoprecipitation, in vitro calcium-induced CAPN5 proteolysis assay, and their cellular fragmentation patterns were compared in parental and CAPN5-deficient SH-SY5Y cells. Here, we provide evidence for CAPN5-mediated proteolysis of the synaptic proteins DLGAP4, IQSEC1 and MPDZ, the neurodegeneration-related EWS, hnRNPU, TFG and UGP2, the DNA replication regulator MCM3, and the neuronal differentiation regulator LMTK1. Our data provide new relevance for neovascular inflammatory vitreoretinopathy (NIV), a progressive eye disease caused by pathogenic mutations in CAPN5. Data are available via ProteomeXchange with identifier PXD064313.", "claims": [{"id": "claim-method", "statement": "An interquartile boxplot analysis [28] was applied to determine whether the identified peptides were at significantly different levels in the two cell lines.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:40650235", "locator": "sections[4] \"2.2. 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Thirty neo-N-termini co", "ere compared in parental and CAPN5-deficient SH-SY5Y cells. Here, we provide evidence for CAPN5-m", "l Culture and the Generation of the CAPN5−/− SH-SY5Y Cell Line The SH-SY5Y human neuroblastoma ce", "ration of the CAPN5−/− SH-SY5Y Cell Line The SH-SY5Y human neuroblastoma cell line (ATCC, Manassa"], "order_basis": "paper_text_order:562", "id": "assay:1"}, {"role": "data_generation", "definition_id": "transfection", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["CO2/95% air at 37 °C. The SH-SY5Y cells were transfected with Calpain-5 Double Nickase CRISPR Plasmid", "c-403881-NIC) using the LipoJet DNA In Vitro Transfection Reagent (Signagen, Frederick, MD, USA, SL100", "Signagen, Frederick, MD, USA, SL100468). 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