Spatiotemporal-Controlled Reporter for Cell-Surface Proteolytic Enzyme Activity Visualization.

Live-cell imaging of cell-surface-associated proteolytic enzymes is crucial to understand their biological roles and functions in both physiological and pathological processes. However, the complexity of the cell membrane environment increases difficulties in specifically investigating targeted proteolytic activities within the microenvironment. Towards this end, a unique, photoremovable, furin-responsive peptide probe that can undergo spatiotemporal control through UV-light illumination has been designed and synthesized to aid in visualizing the activity of a cell-surface-associated protease enzyme, furin, in live cells. Prior to light irradiation, the photolabile moiety, 4,5-dimethoxy-2-nitrobenzyl, in the peptide sequence of the reporter will block furin-like enzymatic hydrolysis, and thus, no fluorescence will be observed. Upon simple light illumination, photolysis will occur, thereby revealing the uncaged peptide probe, which can undergo enzyme hydrolysis and lead to an increase in fluorescence signal; this allows the real-time imaging of endogenous cell-surface-associated furin-like enzyme function in living cells through precise spatial and temporal resolution.