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N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis
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.
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2006 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 348, no 2, 379-385 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2006. Vol. 348, no 2, 379-385 p.
Keyword [en]
Prion protein, N-terminus, Cell-penetrating peptide, Endocytosis, Macropinocytosis, Proteoglycan
National Category
Biological Sciences Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-23006DOI: 10.1016/j.bbrc.2006.07.065OAI: oai:DiVA.org:su-23006DiVA: diva2:189890
Available from: 2006-10-31 Created: 2006-10-31 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Naturally derived cell-penetrating peptides and applications in gene regulation: A study on internalization mechanisms and endosomal escape
Open this publication in new window or tab >>Naturally derived cell-penetrating peptides and applications in gene regulation: A study on internalization mechanisms and endosomal escape
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery.

The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined.

The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2006. 83 p.
Keyword
peptide, oligonucleotide, endocytosis, gene regulation
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-1328 (URN)
Public defence
2006-12-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2006-10-31 Created: 2006-10-31 Last updated: 2010-01-11Bibliographically approved
2. 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
Opponent
Supervisors
Available from: 2007-10-04 Created: 2007-10-04 Last updated: 2017-12-01Bibliographically approved

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Eriksson, L. E. GöranLangel, ÜloAstrid, Gräslund
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