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| 2. |
- Persson, Daniel, 1972, et al.
(författare)
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Penetratin-induced Aggregation and Subsequent Dissociation of Negatively Charged Phospholipid Vesicles
- 2001
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Ingår i: FEBS Letters. - 1873-3468 .- 0014-5793. ; 505:2, s. 307-312
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Tidskriftsartikel (refereegranskat)abstract
- The interaction of the cellular delivery vector penetratin with a model system consisting of negatively charged phospholipid vesicles has been studied. Above a certain peptide to lipid molar ratio, the cationic oligopeptide induces vesicle aggregation. Interestingly, the aggregation is followed by spontaneous disaggregation, which may be related to membrane translocation of the peptide. Circular dichroism (CD) measurements indicate a conformational transition, from alpha -helix to antiparallel beta -pleated sheet, which is simultaneous with the aggregation process. The potential influence of spectroscopic artifacts on CD data due to the drastically increased turbidity during aggregation is discussed.
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| 3. |
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| 4. |
- Persson, Daniel, 1972, et al.
(författare)
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Vesicle size-dependent translocation of penetratin analogs across lipid membranes
- 2004
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 1879-2642 .- 0005-2736. ; 1665:1-2, s. 142-155
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Tidskriftsartikel (refereegranskat)abstract
- The recent discoveries of serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides (CPPs) have prompted a reevaluation of the current understanding of peptide-mediated cellular delivery. Following a report on the differential cellular uptake of a number of penetratin analogs in unfixed cells, we here investigate their membrane translocation abilities in large and giant unilamellar vesicles (LUVs and GUVs, respectively). Surprisingly, in contrast to the behavior in living cells, all peptides readily entered the giant vesicles ( > 1 μm) as proved by confocal microscopy, while none of them could cross the membranes of LUVs (100 nm). For determination of the location of the peptides in the LUVs, a new concept was introduced, based on sensitive resonance energy transfer (RET) measurements of the enhanced fluorescence of acceptor fluorophores present solely in the inner leaflet. An easily adopted method to prepare such asymmetrically labeled liposomes is described. The membrane insertion depths of the tryptophan moieties of the peptides were determined by use of brominated lipids and found to be very similar for all of the peptides studied. We also demonstrate that infrared spectroscopy on the lipid carbonyl stretch vibration peak is a convenient technique to determine phospholipid concentration. © 2004 Elsevier B.V. All rights reserved.
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| 5. |
- Thoren, Per, 1972, et al.
(författare)
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Membrane binding and translocation of cell-penetrating peptides
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 43:12, s. 3471-3489
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R 7 W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of ∼10-11 Å from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.
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| 6. |
- Thoren, Per, 1972, et al.
(författare)
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Membrane destabilizing properties of cell-penetrating peptides
- 2005
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Ingår i: Biophysical Chemistry. - : Elsevier BV. - 1873-4200 .- 0301-4622. ; 114:2-3, s. 169-179
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Tidskriftsartikel (refereegranskat)abstract
- Although cell-penetrating peptides (CPPs), also denoted protein transduction domains (PTDs), have been widely used for intracellular delivery of large and hydrophilic molecules, the mechanism of uptake is still poorly understood. In a recent live cell study of the uptake of penetratin and tryptophan-containing analogues of Tat(48-60) and oligoarginine, denoted TatP59W, TatLysP59W and R7W, respectively, it was found that both endocytotic and non-endocytotic uptake pathways are involved [Thorén et al., Biochem. Biophys. Res. Commun. 307 (2003) 100-107]. Non-endocytotic uptake was only observed for the arginine-rich peptides TatP59W and R7W. In this paper, the interactions of penetratin, R7W, TatP59W and TatLysP59W with phospholipid vesicles are compared in the search for an understanding of the mechanisms for cellular uptake. While R7W, TatP59W and TatLysP59W are found to promote vesicle fusion, indicated by mixing of membrane components, penetratin merely induces vesicle aggregation. Studies of the leakage from dye-loaded vesicles indicate that none of the peptides forms membrane pores and that vesicle fusion is not accompanied by leakage of the aqueous contents of the vesicles. These observations are important for a proper interpretation of future experiments on the interactions of these peptides with model membranes. We suggest that the discovered variations in propensity to destabilize phospholipid bilayers between the peptides investigated, in some cases sufficient to induce fusion, may be related to their different cellular uptake properties.
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| 7. |
- Thoren, Per, 1972, et al.
(författare)
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The Antennapedia peptide penetratin translocates across lipid bilayers - the first direct observation
- 2000
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Ingår i: FEBS Letters. - 1873-3468 .- 0014-5793. ; 482:3, s. 265-268
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Tidskriftsartikel (refereegranskat)abstract
- The potential use of polypeptides and oligonucleotides for therapeutical purposes has been questioned because of their inherently poor cellular uptake. However, the 16-mer oligopeptide penetratin, derived from the homeodomain of Antennapedia, has been reported to enter cells readily via a non-endocytotic and receptor- and transporter-independent pathway, even when conjugated to large hydrophilic molecules. We here present the first study where penetratin is shown to traverse a pure lipid bilayer. The results support the idea that the uptake mechanism involves only the interaction of the peptide with the membrane lipids. Furthermore, we conclude that the translocation does not involve pore formation.
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