Weiss_2025_Life.Sci.Alliance_8_e202403076

Dipeptidyl peptidases 8 and 9 (DPP8/9) are critical for the quality control of mitochondrial and endoplasmic reticulum protein import, immune regulation, cell adhesion, and cell migration. Dysregulation of DPP8/9 is associated with pathologies including tumorigenesis and inflammation. Commonly, DPP8/9 activity is analysed by in vitro assays using artificial substrates, which allow neither continuously monitoring DPP8/9 activity in individual, living cells nor detecting effects from endogenous interactors and posttranslational modifications. Here, we developed DiPAK (for DPP8/9 activity sensor based on AK2), a ratiometric genetically encoded fluorescent sensor, which enables studying DPP8/9 activity in living cells. Using DiPAK, we determined the dynamic range of DPP8/9 activity in cells overexpressing or lacking DPP9. We identified distinct activity levels among melanoma cell lines and found that LPS-induced primary B-cell activation depends on DPP8/9 as the absence of DPP8/9 activity results in apoptotic but not pyroptotic cell death. Consistently, we observed increasing DPP8/9 activity during B-cell maturation. Overall, DiPAK is a versatile tool for real-time single-cell monitoring of DPP8/9 activity in a broad range of cells and organisms.