Bidirectional crosstalk between the catalytic and moonlighting functions of human dipeptidyl peptidase 3: potential physiological implications.

Dipeptidyl peptidase 3 (DPP3) is a ubiquitously expressed zinc-exopeptidase involved in oligopeptide degradation. Beyond its catalytic role, DPP3 exhibits a moonlighting role in the Keap1-Nrf2 signalling pathway, where it promotes Nrf2-dependent gene transcription through an ETGE motif-mediated interaction with the Kelch domain of Keap1. This highlights its role in oxidative stress and cancer, as persistent pathway activation supports tumour cell survival, oxidative stress resistance, and proliferation. While DPP3's catalytic activity is not required for Keap1 binding, the effects of enzyme inactivation on this interaction, and vice versa, remain unclear. Using isothermal titration calorimetry and molecular dynamics simulations, we show that DPP3 inactivation, via mutation (E451A) or inhibitor binding, enhances DPP3-Keap1 complex formation by facilitating detachment of the ETGE-loop in the closed enzyme conformation, a rate-limiting step in complex assembly. Kinetic measurements further reveal that Keap1 binding modulates DPP3 catalytic activity, increasing efficiency through disproportional reductions in both k cat and K m, likely by stabilizing the closed, catalytically competent conformation of the enzyme, which favours substrate binding but hinders the turnover of reaction participants. These findings reveal a bidirectional regulatory mechanism: catalytic inhibitor binding promotes moonlighting interactions with Keap1, while Keap1 binding enhances enzymatic activity. This dual regulation provides insight into how modulation of DPP3's catalytic function could influence Keap1-Nrf2 redox signaling and suggests how cancer cells might exploit multifunctional enzymes like DPP3 to support tumour progression. This hypothesis based on biochemical and computational data remains to be validated in in vitro models.