Cationic cargo and cell-penetrating peptide entry mechanisms

Cell-penetrating peptides (CPPs) are typically short cationic amino acid sequences that can be hooked to a variety of cargos thus allowing their transport into cells for diagnostic, therapeutic, and experimental purposes. Cellular uptake of CPPs occurs through two, not mutually exclusive, modes of entry: direct translocation and endocytosis. We have recently clarified the mechanism of CPP direct translocation by showing that CPPs enter cells through ~2nm-wide water pores created when the plasma membrane is hyperpolarized to around -150 mV. This level of polarization that we have coined "megapolarization" is induced by the combined activity of potassium channels and the positive charges carried by CPPs that accumulate at the cell surface. An important outstanding question is whether natural proteins bearing CPP sequences, such as homeoproteins (HPs), also enter cells via this mechanism.CPPs also employ endocytosis to enter cells. Endocytosis is a major entry route used by cells to take up a variety of extracellular substances. The endocytosed material transits from early endosomes to late endosomes/lysosomes. Even though endocytosis can proceed in a variety of manners, the current evidence indicates that all endocytosed substances follow a single endocytic maturation pathway towards LAMP1-containing lysosomes that is characterized molecularly by the presence of Rab5 on early endosomes and Rab7 on late endosomes. We have discovered that CPPs use a second endosomal pathway en route to non-acidic LAMP1-positive compartment that had escaped detection so far. We have determined that progression along this endocytic pathway requires the Rab14 protein but not Rab5 and Rab7. We have also found that this endocytic route is taken by other, physiological, cationic cargos such as polyamines or homeodomains (HDs). We have now designed a series of screens to identify proteins involved at different stages of this new Rab14-dependent endocytic pathway.While it is generally assumed that cargos linked to CPPs escape endosomes in order to exert their biological activity, little is known about the nature of the endosomal escape mechanism. Of note, studies on endosomal escape are potentially confounded by the direct translocation abilities of CPPs. Thus, assessing endosomal escape should be done in conditions where direct translocation is inhibited to exclude the possibility that cytosolic acquisition of CPPs is a consequence of direct entry through the plasma membrane. Consequently, we propose to develop an assay that allows studying endosomal escape in the absence of direct translocation. Such assay will then be used to assess the endosomal escape properties of specific CPPs and will also be used to screen chemical libraries for compounds promoting CPP endosomal escape.This grant proposal will address the following aims:Aim 1. Characterize at the biochemical, molecular, and functional levels the Rab14-dependent endocytic pathway taken by CPPs and other cationic materialAim 2. Investigate how HPs enter cellsAim 3. Establish an experimental setting that limits the process of CPP direct translocation to specifically assess the endosomal escape properties of CPPs and to discover small molecules that favor CPP endosomal escape