Visualization of Ectopic Serine Protease Activity by Förster Resonance Energy Transfer-Based Reporters

Channel-activating proteases (CAPs) play a fundamental role in the regulation of sodium transport across epithelial tissues mainly via cleavage-mediated fine-tuning of the activity of the epithelial sodium channel (ENaC). Hyperactivity of CAPs and subsequently increased ENaC activity have been associated with various diseases, including cystic fibrosis (CF). To date, there is only a limited number of tools available to investigate CAP activity. Here, we developed ratiometric, peptide-based Förster resonance energy transfer (FRET) reporters useful to visualize and quantify the activity of ectopic serine proteases including the CAPs prostasin and matriptase in human and murine samples in a temporally and spatially resolved manner. Lipidated varieties were inserted into the outer leaflet of the plasma membrane to detect enzyme activity on the surface of individual cells, that is, close to the protease substrates. The FRET reporters (termed CAPRee) selectively detected the activity of ectopic serine proteases such as CAPs in solution and on the surface of human and murine cells. We found increased CAP activity on the surface of cells with a genetic background of CF. The new reporters will contribute to a better understanding of ectopic serine protease activity and their regulation under physiological and pathophysiological conditions.