Trivedi_2025_FEBS.Lett__

Dipeptidyl peptidase IV (DPPIV) family proteases are classically defined by their strict removal of N-terminal dipeptides from substrates bearing a proline or alanine at the P(1) position. Here, we report that both Caenorhabditis elegans DPF-3 and human DPP4 (hDPP4) possess previously unrecognized tripeptidyl peptidase activity in addition to dipeptidyl peptidase activity. This activity plays a key role in the processing of the WAGO-1 protein N-terminus, which is essential for proper small-RNA loading, germline genome defense, and fertility. Kinetic analyses using the fluorogenic substrate H-Met-Gly-Pro-AMC further demonstrated that, in vitro, DPF-3 and hDPP4 can liberate AMC. These findings potentially expand the substrate repertoire of DPPIV proteases, suggesting that these proteases could function as versatile N-terminal processors, with important implications for nascent protein maturation.