The Caenorhabditis elegans DPF ‐3 and human DPP4 have tripeptidyl peptidase activity

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.