Membrane Binding and Translocation of Cell-Penetrating Peptides

Abstract

Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R(7)W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of approximately 10-11 A from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.

Cell-penetrating and cell-targeting peptides in drug delivery

“…It was first proposed that CPPs, especially Tat and Antennapedia, but also others such as poly-Arg [10,26], Transportan [27], MPG [28] or Pep-1 [20], could pass through the plasma membrane via an energy-independent pathway. Some suggestions have been put forward to explain the translocation of these peptides, such as the formation of micromicelles at the membrane [5], or direct translocation through the lipid bilayer [29,30]. If conceivable for small CPPs, these models can not explain the passage through the plasma membrane of CPPs-cargoes of very important size [31,32].…”

Topography Studies on the Membrane Interaction Mechanism of the Eosinophil Cationic Protein

“…The degree of membrane penetration of ECP was followed by analyzing the quenching effect of brominated lipids on both W10 and W35 fluorescence. The synthesized brominated PC derivatives, which do not contain double bonds, form bilayers of equal thickness and properties similar to lipids with one unsaturated bond, because bromine is a small molecule that does not drastically disturb the lipid bilayer (49), and the bulkiness of the bromine atoms has similar effects on lipid packing as a cis double bond (50).…”

Label-free probe of HIV-1 TAT peptide binding to mimetic membranes

“…Among the techniques are isothermal titration calorimetry (9,11), fluorescence spectroscopy (12,13), FRET (12,14), single-molecule fluorescence microscopy (15,16), and solid-state NMR (17). Second harmonic generation (SHG), as an interface-selective technique (18)(19)(20)(21)(22)(23)(24), does not require a label, and because SHG is sensitive to the interface potential, it is an attractive method to selectively probe the binding of the highly charged (+8) TAT peptide to liposome surfaces.…”