Abstract
Cationic cell-penetrating peptides (CPPs) have been used widely as delivery vectors for the import of molecules that otherwise do not cross the plasma membrane of eukaryotic cells. In this work, we demonstrate that the three cationic CPPs, Antennapedia homeodomain-derived peptide (Antp), nona-arginine and Tat-derived peptide, inhibit tumour necrosis factor (TNF)-mediated signal transduction. This inhibition is based on the downregulation of TNF receptors at the cell surface by induction of internalization. In contrast to TNF-dependent receptor internalization, no receptor activation occurs. The receptor downregulation is not restricted to the CPPs. Remarkably, the HIV-1 Tat protein itself also induces the internalization of TNF receptors. The dynamin dependence of the internalization, as well as the fact that epidermal growth factor receptors are also internalized, suggest a general induction of clathrin-dependent endocytosis as the mechanism of action. The significance of these findings for the use of cationic CPPs in the import of bioactive peptides is demonstrated here using a conjugate consisting of Antp and a Smac protein-derived cargo peptide. The cargo alone, when introduced into cells by electroporation, enhanced TNF-induced apoptosis by inhibiting the anti-apoptotic action of IAPs (inhibitor of apoptosis proteins). For the Antp-Smac conjugate at concentrations below 40 muM the inhibitory effect of the Antp peptide compensated for the pro-apoptotic activity of the cargo, and led to the protection of cells against TNF-mediated apoptosis. These data provide important new information for the use of cationic CPPs for the cellular delivery of bioactive molecules.
Highlights
The import of membrane-impermeable peptides and nucleotides into mammalian cells is becoming increasingly used for the selective interference with cellular signal transduction
We demonstrate that the three cationic cell-penetrating peptides (CPPs), Antennapedia homeodomain-derived peptide (Antp), nona-arginine and Tat-derived peptide, inhibit tumour necrosis factor (TNF)-mediated signal transduction
The human immunodeficiency virus-1 (HIV-1) Tat protein itself induces the internalization of TNF receptors
Summary
The import of membrane-impermeable peptides and nucleotides into mammalian cells is becoming increasingly used for the selective interference with cellular signal transduction. The import strategies presented so far can be subdivided into those that transiently permeabilize the plasma membrane, such as electroporation and streptolysine O treatment, and those employing carrier molecules that enter the cell while maintaining membrane integrity (Stephens and Pepperkok, 2001). Among the latter, cell-penetrating peptides (CPPs) have become established as a powerful tool for a noninvasive cellular delivery of peptides, oligonucleotides and even nanoparticles (Langel, 2002; Fischer et al, 2001). It has been shown (i) that translocation of CPPs from vesicular compartments into the cytoplasm requires acidification of endosomes (Fischer et al, 2004; Potocky et al, 2003) and (ii) that retrograde transport may be involved in the trafficking and release into the cytoplasm (Fischer et al, 2004)
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