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Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport Across Phospholipid Membranes Induced by a Proton Gradient
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2011 (English)In: Journal of drug delivery, ISSN 2090-3022, Vol. 2011, 897592- p.Article in journal (Refereed) Published
Abstract [en]

Detergent-mediated reconstitution of bacteriorhodopsin (BR) into large unilamellar vesicles (LUVs) was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG) as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

Place, publisher, year, edition, pages
2011. Vol. 2011, 897592- p.
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-66760DOI: 10.1155/2011/897592OAI: oai:DiVA.org:su-66760DiVA: diva2:468492
Available from: 2011-12-21 Created: 2011-12-21 Last updated: 2011-12-28Bibliographically approved
In thesis
1. Biophysical studies of peptides with functions in biotechnology and biology
Open this publication in new window or tab >>Biophysical studies of peptides with functions in biotechnology and biology
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

My thesis concerns spectroscopic studies (NMR, CD and fluorescence) of peptides with functions in biotechnology and biology, and their interactions with a model membrane (large unilamellar phospholipid vesicles).

The resorufin-based arsenical hairpin binder (ReAsH) bound to a short peptide is a useful fluorescent tag for genetic labeling of proteins in living cells. A hairpin structure with some resemblance to type II β-turn was determined by NMR structure calculations (Paper I).

Cell-penetrating peptides (CPPs) are short (30-35 residues), often rich in basic amino acids such as Arg. They can pass through the cell membrane and deliver bioactive cargoes, making them useful for biotechnical and pharmacological applications. The mechanisms of cellular uptake and membrane translocation are under debate. Understanding the mechanistic aspects of CPPs is the major focus of Papers II, III, and IV.

The effect of the pyrenebutyrate (PB) on the cellular uptake, membrane translocation and perturbation of several CPPs from different subgroups was investigated (Paper II). We concluded that both charge and hydrophobicity of the CPP affect the cellular uptake and membrane translocation efficiency.

Endosomal escape is a crucial challenge for the CPP applications. We modeled the endosome and endosomal escape for different CPPs to investigate the corresponding molecular mechanisms (Papers III and IV). Hydrophobic CPPs were able to translocate across the model membrane in the presence of a pH gradient, produced by bacteriorhodopsin proton pumping, whereas a smaller effect was observed for hydrophilic CPPs.

Dynorphin A (Dyn A) peptide mutations are associated with neurodegenerative disorders, without involvement of the opioid receptors. The non-opioid activities of Dyn A may involve membrane perturbations. Model membrane-perturbations by three Dyn A mutants were investigated (Paper V). The results showed effects to different degrees largely in accordance with their neurotoxic effects.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2012. 75 p.
Keyword
Genetic fluorescence label, Biarsenical tetracysteine motif, Cell-penetrating peptides, Large unilamellar vesicles, Pyrenebutyrate, Endosomal escape, Membrane perturbation, Bacteriorhodopsin, Dynorphin
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-66948 (URN)978-91-7447-417-6 (ISBN)
Public defence
2012-02-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2012-01-23 Created: 2011-12-22 Last updated: 2013-04-09Bibliographically approved

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