| 1. |
- Esbjörner, Elin K., et al.
(author)
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Membrane binding of pH-sensitive Influenza fusion peptides. Positioning, configuration and induced leakage in lipid vesicles models
- 2007
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:47, s. 13490-13504
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Journal article (peer-reviewed)abstract
- pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA 1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion (∼60-65° relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue
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| 2. |
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| 4. |
- Magzoub, Mazin, et al.
(author)
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N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis
- 2006
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In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 348:2, s. 379-385
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Journal article (peer-reviewed)abstract
- A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1–24) and a basic domain (KKRPKP, residues 25–30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp–DNA–gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide’s ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.
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| 5. |
- Oglęcka, Kamila, 1977-
(author)
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Biophysical studies of membrane interacting peptides derived from viral and Prion proteins
- 2007
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Doctoral thesis (other academic/artistic)abstract
- This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.
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| 7. |
- Oglęcka, Kamila, et al.
(author)
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Relevance of the N-terminal NLS-like sequence for membrane interactions of the Prion protein
- 2007
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In: Biochimica et Biophysica Acta. MR. Reviews on Biomembranes. - : Elsevier BV. - 0304-4157 .- 1879-257X. ; 1778:1, s. 206-213
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Journal article (peer-reviewed)abstract
- We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23–28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1–28 (mPrPp(1–28)) and 23–50 (mPrPp(23–50)), respectively. In erythrocytes, mPrPp(1–28) induced 60% haemoglobin leakage after 30 min, whereas mPrPp(23–50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be 12% for 50 μM mPrPp(1–28), and 1% for 50 μM mPrPp(23–50). Circular dichroism spectra showed structure induction of mPrPp(1–28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mPrPp(23–50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(1–28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23–50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.
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| 8. |
- Oglęcka, Kamila, et al.
(author)
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Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects
- 2008
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2736. ; 1778:1, s. 206-213
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Journal article (peer-reviewed)abstract
- We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23-28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1-28 (mPrPp(1-28)) and 2350 (rnPrPp(23-50)), respectively. In erythrocytes, mPrPp(1-28) induced similar to 60% haemoglobin leakage after 30 min, whereas mprPp(23-50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be similar to 12% for 50 mu M mPrPp(1-28), and similar to 1% for 50 mu M mPrPp(23-50). Circular dichroism spectra showed structure induction of mPrPp(1-28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mprPp(23-50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(I-28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23-50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.
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