Delivery of proteins in live cells with viral peptides: principles and mechanisms

Cell-penetrating peptides (CPPs) mediate the delivery of macromolecules across the plasma membrane of live cells. These peptides are therefore important due to the potential of making the delivery of protein probes or therapeutics a routine procedure. However, CPP-mediated delivery is currently an inefficient process. CPP-protein conjugates are internalized into cells by endocytosis and the macromolecules remain trapped inside endosomes instead of reaching the target cellular localization. To solve this problem, we report a delivery methodology which relies on the use of a chimera of the TAT and of the Influenza HA2. TAT is a prototypical CPP that can promote macropinocytosis in live cells and HA2 is a pH-sensitive peptide that destabilizes lipid membranes upon acidification. I demonstrate that HA2-TAT can deliver a variety of macromolecular cargos into live mammalian cells by a simple co-incubation protocol. A model is described where TAT causes the endocytic uptake of cargos present in the media and that HA2 disrupts the endosomal membrane upon endosomal acidification. In addition, using red blood cells as a model system, HA2-TAT binds to membranes in a pH-dependent manner and causes the formation of pores through which macromolecules can diffuse. Additionally, the pro-apoptotic domain (PAD) peptide is also successfully delivered by HA2-TAT and shows significant apoptosis in cells through macropinocytosis.