Penetratin and Related Cell-Penetrating Cationic Peptides Can Translocate Across Lipid Bilayers in the Presence of a Transbilayer Potential

Abstract

Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH(2) and K(6)GC-NH(2), are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.

Translocation of Dynorphin Neuropeptides across the Plasma Membrane

“…Consistently with these predictions substance P, R 7 W, Tat 47-57, and the signal sequence hydrophobic region peptides have been demonstrated to translocate into live cells at 4°C or in the presence of metabolic inhibitors (22,(57)(58)(59). CPP translocation across model lipid membranes also supports this notion (60,61). Models of the second type consider clathrin-mediated endocytosis, caveolar endocytosis, and lipid raft-dependent macropinocytosis as translocation mechanisms (8 -10, 57).…”

Evaluation of Cell-Penetrating Peptide/Adenovirus Particles for Transduction of CAR-Negative Cells

“…These studies demonstrated that transmembrane potential is required for the peptides to be absorbed by neutral and charged lipid bilayer systems (Terrone, Sang et al 2003;Ziegler, Blatter et al 2003;.…”

NMR solution structure and position of transportan in neutral phospholipid bicelles

“…Both the galanin and mastoparan parts of transportan display secondary H a shifts characteristic of a helical structure, and the H N secondary shifts for the mastoparan part varies with a periodicity of three to four amino acid residues, indicative of an amphipathic helix. The structure of transportan was calculated based on 220 proton-proton distances shorter than 6 A as determined from NOESY cross-peaks (NOEs)-derived distance constraints. The final solution structure of transportan is seen to adopt a helical conformation between residues Asn15 and Ile26 (Fig.…”

“…In recent studies it was shown that fluorescence microscopy on fixed cells, which is a method by which several translocation observations have been made, can give artifactual uptake of peptide associated with the plasma membrane, and that endocytotic pathways may dominate for many systems [3][4][5]. Other translocation mechanisms have also been proposed [6,7], and since the mechanism of cell internalization is not fully known, further work is needed in order to clarify whether the internalization is dependent on the structure of the peptide or on the orientation of the peptide in relation to the membrane.…”