Cell-penetrating Peptides

Abstract

Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48 -60 and (Arg) 9 . We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.During the last decade, several proteins, such as HIV-1 Tat, Drosophila Antennapedia homeoprotein, and HSV-1 VP22 have been shown to traverse the cell membrane by a process called protein transduction and to reach the nucleus while retaining their biological activity (1-5). Short "protein-transduction domains" are responsible for the cellular uptake of these proteins (6, 7). Although the biological relevance of protein transduction remains to be understood, it has attracted much interest. Indeed, it was discovered that short peptides derived from protein-transduction domains (cell-penetrating peptides or CPPs) 1 can be internalized in most cell types and, more importantly, allow the cellular delivery of conjugated (or fused) biomolecules (8, 9). A wide range of biomolecules such as antigenic peptides (10) The mechanism of internalization of CPPs and their cargo is not well understood and has recently been the subject of controversies. It has been described in the literature that internalization of these peptides is not significantly inhibited by incubation at low temperature, by depletion of the cellular ATP pool, or by inhibitors of endocytosis (7, 20 -22). Moreover, structure-activity studies indicate that the internalization of CPPs do not depend on its specific primary sequence, which implies independence of receptor recognition (20,23,24). Based on these results, it has been commonly accepted that the internalization of CPPs do not involve endocytosis or specific protein transporters. Instead, a direct transport through the lipid bilayer of membranes has been proposed as a possible mechanism of translocation (7,20). If correct, this mechanism would require a radical revision of current ideas on the properties of lipid bilayers, taking into account the hydrophilic nature of CPPs such as Tat or (Arg) 9 and the fact that there is no indication of increased membrane permeability in the p...

A Stepwise Dissection of the Intracellular Fate of Cationic Cell-penetrating Peptides

“…The distortion of uptake experiments by peptides that were only associated with the plasma membrane but had not been internalized by the cells was avoided by treating the cells with trypsin prior to flow cytometry (29). While Fluo-R9 and FluoAntp were taken up efficiently to comparable levels, the intracellular fluorescence of Fluo-Tat was less than 10% of that of cells treated with the other two peptides (Fig.…”

“…, and Tat Peptides-Considering two recent studies demonstrating the involvement of endocytosis in the uptake of the R9 peptide, the HIV-1 Tat peptide (29), and Tat fusion proteins (30), we intended to dissect in detail the cellular mechanisms involved in the uptake of cationic CPPs (Table I) and their release into the cytosol. Initially the effect of wortmannin, bafilomycin A1, and chloroquine as inhibitors of endocytosis and endosomal acidification on the uptake of fluorescently labeled CPPs was assessed by flow cytometry of living cells.…”

“…HeLa cells were chosen as a second cell line because in the study of Richard et al (29) this cell line was also investigated. In contrast to MC57 cells (Fig.…”

“…Endocytosis is clearly involved in the internalization of the HIV-1 Tat peptide (29). For Tat fusion proteins it was demonstrated that cellular internalization occurs through a temperature-dependent endocytic pathway that originates from lipid rafts and follows caveolar endocytosis (30).…”

Tuning the Properties of Penetrating Peptides by Oxime Conjugation

“…10 Initial passive diffusion assumptions have been ruled out because of artifacts related with the cytoplasm distribution after cell fixation. 11 Currently, it is widely accepted that penetrating peptides can be transported by several mechanisms operating at the same time. Additionally, the final internalization pathway of a given peptide is often promiscuous and depends on different parameters such as concentration, nature of the cargo and size of the corresponding supramolecular aggregates.…”