A doubly labeled penetratin analogue as a ratiometric sensor for intracellular proteolytic stability.

Endocytosis has been shown to play a major role in the cellular import of cationic cell-penetrating peptides (CPPs) and CPP conjugates. Considering the presence of proteolytic activities inside the endolysosomal compartment, it is necessary to assess the consequences of the import mechanism on the intracellular integrity of the vector and the cargo. In this work, a penetratin analogue terminally labeled with two different fluorophores was synthesized and used as a sensor to quantitatively dissect the contribution of intracellular proteolytic activities on the breakdown of this specific CPP. Using a panel of lysosomal protease inhibitors, the endocytic compartment was identified as the major site of degradation. In contrast, an inhibitor of the proteasome had little effect on intracellular peptide integrity. Very remarkably, inhibitors of endolysosomal proteolysis also affected the intracellular distribution of fluorescence, leading to a reduction of fluorescein fluorescence in the cytoplasm. This change in fluorescence distribution was very similar to the one observed after incubation of cells with inhibitors of endosomal acidification. These results indicate that cytoplasmic fluorescence, typically interpreted as CPP entering the cytosol, may originate from proteolytic breakdown products.