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Relevance of the N-terminal NLS-like sequence for membrane interactions of the Prion protein
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2007 (English)In: Biochimica et Biophysica Acta. MR. Reviews on Biomembranes, ISSN 0304-4157, E-ISSN 1879-257X, Vol. 1778, no 1, 206-213 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2007. Vol. 1778, no 1, 206-213 p.
Keyword [en]
Prion protein, Membrane translocation, Calcein leakage, Endosomal escape, NLS-like sequence, Haemoglobin leakage
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:su:diva-24501DOI: 10.1016/j.bbamem.2007.09.034OAI: oai:DiVA.org:su-24501DiVA: diva2:197661
Note

Part of urn:nbn:se:su:diva-7109

Available from: 2007-10-04 Created: 2007-10-04 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Biophysical studies of membrane interacting peptides derived from viral and Prion proteins
Open this publication in new window or tab >>Biophysical studies of membrane interacting peptides derived from viral and Prion proteins
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2007. 69 p.
Keyword
Prion peptides, Doppel peptide, Influenza fusion peptides, peptide-membrane interactions, translocation, linear dichroism, circular dichroism
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-7109 (URN)978-91-7155-502-1 (ISBN)
Public defence
2007-10-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
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Available from: 2007-10-04 Created: 2007-10-04 Last updated: 2017-12-01Bibliographically approved

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Eriksson, L. E. GöranLangel, ÜloGräslund, Astrid
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