Consequences of nonlytic membrane perturbation to the translocation of the cell penetrating peptide pep-1 in lipidic vesicles.
The action of the cell penetrating pep-1 at the molecular level is not clearly understood. The ability of the peptide to induce (1) vesicle aggregation, (2) lipidic fusion, (3) anionic lipid segregation, (4) pore or other lytic structure formation, (5) asymmetric lipidic flip-flop, and (6) peptide translocation across the bilayers in large unilamellar vesicles was studied using photophysical methodologies mainly related to fluorescence spectroscopy. Neflometry and turbidimetry techniques show that clustering of vesicles occurs in the presence of the peptide in a concentration- and anionic lipid content-dependent manner. Results from Forstër resonance energy transfer-based methodologies prove lipidic fusion and anionic lipid segregation, but no evidence for pores or other lytic structures was found. Asymmetric lipid flip-flop was not detected either. A specific method related to the quenching of the rhodamine-labeled lipids by pep-1 was developed to study the eventual translocation of the peptide. Translocation does not occur in symmetrical neutral and negatively charged vesicles, except when a valinomycin-induced transmembrane potential exists. Our work strongly suggests that the main driving force for peptide translocation is charge asymmetry between the outer and inner leaflet of biological membranes and reveals that pep-1 is able to perturb membranes without being cytotoxic. This nonlytic perturbation is probably mandatory for translocation to occur.
Centro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Ed. C8, Campo Grande, 1749-016 Lisboa, Portugal.
Sónia Troeira
ST
Henriques
Miguel Augusto Rico Botas
MA
Castanho
D000838:Anions / Q000737:chemistry; D002463:Cell Membrane Permeability; D031541:Fluorescence Resonance Energy Transfer; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008561:Membrane Fusion; D008564:Membrane Potentials; D008956:Models, Chemical; D010455:Peptides / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D011485:Protein Binding; D021381:Protein Transport; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity
D000838:Anions; D008051:Lipid Bilayers; D010455:Peptides; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; C028694:1-palmitoyl-2-oleoylphosphatidylcholine