Cationic cell-penetrating peptides induce ceramide formation via acid sphingomyelinase: implications for uptake.
Cationic cell-penetrating peptides (CPP) are receiving increasing attention as molecular transporters of membrane-impermeable molecules. Import of cationic CPP occurs both via endocytosis and - at higher peptide concentrations - in an endocytosis-independent manner via localized regions of the plasma membrane. At present, this endocytosis-independent import of cationic CPP is not well understood, but has been shown to be sensitive to various pharmacological inhibitors, suggesting a role of an unidentified enzymatic activity. Here, we demonstrate that the direct translocation of cationic CPP depends on a CPP-induced translocation of acid sphingomyelinase (ASMase) to the outer leaflet of the plasma membrane and ceramide formation. The involvement of ASMase in uptake was confirmed by a pharmacological inhibition of ASMase by imipramine and a subsequent rescue of uptake through external addition of sphingomyelinase, and by using ASMase-deficient cells. We also found that the threshold for direct CPP translocation can be lowered through addition of sphingomyelinase and that sphingomyelinase enhances the translocation of R9 coupled to low-molecular weight cargos, but not high-molecular weight cargos. In conclusion, we show that a previously poorly understood mechanism of cationic CPP import depends on the ASMase-dependent formation of ceramide on the outer leaflet of the plasma membrane. To our knowledge, this is the first illustration that a class of delivery vectors operates through the induction of an enzymatic activity that changes the lipid composition of the plasma membrane.
Verdurmen
Wouter P R
WP
Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.
Thanos
Melissa
M
Ruttekolk
Ivo R
IR
Gulbins
Erich
E
Brock
Roland
R
D000818:Animals; D002412:Cations; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D002518:Ceramides / Q000378:metabolism*; D004337:Drug Carriers / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D002412:Cations; D057846:Cell-Penetrating Peptides; D002518:Ceramides; D004337:Drug Carriers; D004791:Enzyme Inhibitors; D013108:Sphingomyelin Phosphodiesterase