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Interactions of the Cell-Penetrating Peptide Penetratin with Model Membranes and Live Cells

Daniel Persson (Institutionen för kemi och biovetenskap)
Göteborg : Chalmers University of Technology, 2003. ISBN: 91-7291-336-3.

The delivery of large, hydrophilic molecules, such as proteins and oligonucleotides, to the cytoplasm and nucleus of cells is problematic due to their generally low plasma membrane permeability. With the identification of a number of cell-penetrating peptides, capable of mediating non-endocytotic, intracellular delivery of macromolecular cargos, this obstacle now seems surmountable. In this thesis, the interactions of the cell-penetrating peptide penetratin, derived from the homeodomain of the Drosophila transcription factor Antennapedia, with lipid vesicles and live cells were studied.

In accordance with early work on penetratin indicating a receptor- and transporter-independent cellular uptake, penetratin was shown to traverse the membranes of giant unilamellar vesicles, providing evidence that translocation across pure lipid bilayers is indeed possible. However, contrasting results obtained for large unilamellar vesicles demonstrated that the ability to translocate depends on macroscopic properties of the membrane as well. It was also shown that penetratin, due to its high positive charge, has a strong affinity for negatively charged vesicles, and at high peptide to lipid ratios induces vesicles aggregation, which in turn drives an a-helix to β-sheet conformational transition.

In contrast to previous studies of cell internalization of penetratin using fixed or permeabilized cells, uptake in live cells was shown to be both energy-dependent and endocytotic. Also, the two tryptophans in the peptide sequence, earlier considered essential for internalization were found to be dispensable. Instead, arginines were found to be crucial both for cell association and for the mechanism of uptake. Vesicle studies indicated that the cell interaction properties provided by arginines in the peptide sequence are attributable to interactions with components other than the plasma membrane lipids.

During this work, a number of methods with general applicability have been developed. Examples are modification of silica surfaces to prevent adsorption of cationic peptides, a novel analysis to assess peptide binding to liposomes and a method to determine the membrane location of vesicle-associated compounds.

Nyckelord: antennapedia homeodomain, membrane translocation, penetratin, uptake, delivery vector, confocal microscopy, cell-penetrating peptide, protein transduction, antisense, vesicle

Denna post skapades 2006-08-29. Senast ändrad 2013-09-25.
CPL Pubid: 66


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