biostudies-literatureUnknownAygun ISwiss National Science FoundationNIH HHSProteases are enzymes that catalyse the hydrolysis of peptide bonds in proteins for their functional, modification or degradation. Members of the Dipeptidyl Peptidase IV (DPPIV) family are exopeptidases that cleave dipeptides off the N-termini of their substrate peptides, typically after proline or alanine. Recently, we showed that human DPP4 and Caenorhabditis elegans DPF-3 have a larger target repertoire in vitro, permitting cleavage after additional amino acids. Here, we use TAILS (Terminal Amine Isotopic Labelling of Substrates) to identify DPF-3 targets in vivo and observe cleavage of MEP-1 after threonine, confirming a broader substrate specificity of DPF-3 also in vivo. Demonstrating physiological relevance, we show that rendering MEP-1 resistant to cleavbioRxiv : the preprint server for biology2025.09.26.678732https://www.ebi.ac.uk/biostudies/studies/S-EPMC12485953biostudies-literatureCleavage of MEP-1 by DPF-3 Reveals Novel Substrate Specificity and Its Impact on Reproductive Fitness.PMC12485953P40 OD010440182880Aygun ISoneson CHess DGudipati RKSeebacher JGroßhans HAmanullah AfalseCleavage of MEP-1 by DPF-3 Reveals Novel Substrate Specificity and Its Impact on Reproductive Fitness.Proteases are enzymes that catalyse the hydrolysis of peptide bonds in proteins for their functional, modification or degradation. Members of the Dipeptidyl Peptidase IV (DPPIV) family are exopeptidases that cleave dipeptides off the N-termini of their substrate peptides, typically after proline or alanine. Recently, we showed that human DPP4 and Caenorhabditis elegans DPF-3 have a larger target repertoire in vitro, permitting cleavage after additional amino acids. Here, we use TAILS (Terminal Amine Isotopic Labelling of Substrates) to identify DPF-3 targets in vivo and observe cleavage of MEP-1 after threonine, confirming a broader substrate specificity of DPF-3 also in vivo. Demonstrating physiological relevance, we show that rendering MEP-1 resistant to cleav2025-01-01T00:00:00Z2025 Sep2026-06-14T03:21:25.645Z2026-06-14T03:09:03.066ZS-EPMC124859534104032210.1101/2025.09.26.678732