Cellular uptake of Antennapedia Penetratin peptides is a two-step process in which phase transfer precedes a tryptophan-dependent translocation

Abstract

Several homeodomains and homeodomain-containing proteins enter live cells through a receptor- and energy-independent mechanism. Translocation through biological membranes is conferred by the third alpha-helix of the homeodomain, also known as Penetratin. Biophysical studies demonstrate that entry of Penetratin into cells requires its binding to surface lipids but that binding and translocation are differentially affected by modifications of some physico-chemical properties of the peptide, like helical amphipathicity or net charge. This suggests that the plasma membrane lipid composition affects the internalization of Penetratin and that internalization requires both lipid binding and other specific properties. Using a phase transfer assay, it is shown that negatively charged lipids promote the transfer of Penetratin from a hydrophilic into a hydrophobic environment, probably through charge neutralization. Accordingly, transfer into a hydrophobic milieu can also be obtained in the absence of negatively charged lipids, by the addition of DNA oligonucleotides. Strikingly, phase transfer by charge neutralization was also observed with a variant peptide of same charge and hydrophobicity in which the tryptophan at position 6 was replaced by a phenylalanine. However, Penetratin, but not its mutant version, is internalized by live cells. This underscores that charge neutralization and phase transfer represent only a first step in the internalization process and that further crossing of a biological membrane necessitates the critical tryptophan residue at position 6.

Designer peptide delivery systems for gene therapy

“…To what degree this amphipathic quality dictates the route of internalization is not clear. However, complete loss of function of Penetratin was observed following substitution of tryptophan (W6) for phenylalanine (46) . It was then postulated that Penetratin followed a two-step model for internalization that involved initial electrostatic interaction followed by tryptophan-dependent membrane destabilization .…”

Cell‐penetrating peptides: Nanocarrier for macromolecule delivery in living cells

“…A dimmer of the Tat molecule (Tat 2 , RKK RRQRRRRKKRRQRRR) has an increased ability to translocate across animal and plant cell membranes because of the presence of high number of arginine residues (5, 13). The homeodomain of the Drosophila-antennapedia transcription factor (ATF) consists of three alpha-helices with one beta-turn between helices two and three, which binds DNA through a 60 amino acid sequence (14). The truncated N-terminal ATF peptide consists of a 16-residue peptide (RQIKIWFQNRRMKWKK) also referred to as penetratin, which is capable of translocation across cell membranes (15,16).…”

LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein

“…Genetically normal rat and mouse neurons endocytosed their endogenous PrP C at the same rate and were inhibited by the siRNA LRP1 used with similar efficiency. For siRNA delivery by penetratin-1 (Davidson et al, 2004;Dom et al, 2003), all extracellular DNA was removed by treating cultures overnight with RNAse-free DNAse (5 units/ml; Qiagen) and including the enzyme in the incubation with siRNApenetratin.…”