Study on the internalization mechanism of the ZEBRA cell penetrating peptide

Cell-penetrating peptides (CPPs) represent a noninvasive method for delivering functional biomolecules into living cells. We have recently shown that the Epstein-Barr virus transcriptional factor ZEBRA contains a protein transduction domain, named Z9 or minimal domain (MD). Only few of currently identified CPPs including MD are able to rapidly cross the mammalian cell membrane without being entrapped into endosomal compartments, even when fused to cargo macromolecules. In this work, a series of MD deletion mutants has been engineered and their cellular uptake has been analyzed by confocal microscopy and FACS. We identified a domain MD 11 (8 amino acids shorter than MD) able to enter mammalian cells via a mainly endocytosis-independent mechanism. All the other generated truncated forms exhibited reduced cellular uptake and penetrated into cells through endocytic mechanisms. These results have highlighted the role of the MD 11 C-terminal region as essential for efficient cellular entry and endosomal escape  and open new perspectives for the use of this CPP as carrier for delivering biologically active macromolecules with therapeutic potential.