Cleavage of MEP-1 by DPF-3 reveals novel substrate specificity and its impact on reproductive fitness.

Proteases are enzymes that catalyze 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 terminal amine isotopic labeling of substrates (TAILS) 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 cleavage disrupts its stability, leading to developmental abnormalities such as defective gonadal migration and reproductive issues. Collectively, our findings highlight a previously unappreciated complexity in the substrate specificity of DPPIV family proteases and suggest that their physiological roles may extend beyond what is currently known.