Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide–membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S413-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S413-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S413-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S413-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S413-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S413-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide–membrane interactions.