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Cell-penetrating peptides in model membrane systems: Interaction, structure induction and membrane effects
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Despite continuing advances in the development of macromolecules, including peptides, proteins, and oligonucleotides, for therapeutic purposes, the successful application of these hydrophilic molecules has so far been hampered by their inability to efficiently traverse the plasma membrane. The discovery of a class of peptides (cell-penetrating peptides, CPPs) with the ability to mediate the non-invasive and efficient import of a whole host of cargoes, both in vitro and in vivo, has provided a new means by which the problem associated with cellular delivery can be circumvented.

A complete understanding of the translocation mechanism(s) of CPPs has so far proven elusive. Initial studies indicated an ATP-independent, non-endocytotic mechanism, dependent on direct peptide-membrane interactions, making it an enticing challenge from a biophysical point of view. To gain an insight into this mechanism, we identified three new CPP sequences, one corresponding to the third helix of the Islet-1 homeodomain, the other two corresponding to the unprocessed N-termini of the mouse and bovine PrPs, denoted mPrPp and bPrPp, respectively. We then investigated the membrane interactions of these peptides, comparing them to two well-characterized CPPs, the Antennapedia homeodomain-derived pAntp, and the chimeric transportan, in a variety of model membrane systems, using several spectroscopic techniques.

Both pAntp and transportan were found to reside in the headgroup region of the bilayer, oriented along the surface (perpendicular to the bilayer normal). However, differences were observed between the peptides – with the homeodomain-derived peptides, pAntp and pIsl, on the one hand, and transportan and the prion-derived peptides on the other – in terms of their membrane interactions, in particular their membrane perturbation effects. These differences suggest that the peptides belong to two classes of CPPs that translocate through different mechanisms. This hypothesis was given further substance by the recent re-evaluation of the translocation mechanism, which led to the conclusion that many peptides, including pAntp, translocate by an energy-dependent, endocytotic mechanism.

Interesting structural behaviour was observed for the homeodomain-derived CPPs, where they readily underwent an α → β structural conversion, depending on experimental conditions. High peptide concentration and/or high negative membrane surface charge was found to promote β-sheet structure. This structural conversion characteristic was shared by the prion-derived peptides, which along with their CPP property and their membrane perturbation effects, may have implications for the infectivity and toxicity associated with prion diseases.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik , 2004. , 68 p.
National Category
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-247ISBN: 91-7265-956-4 (print)OAI: oai:DiVA.org:su-247DiVA: diva2:191482
Public defence
2004-10-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 14:00
Opponent
Supervisors
Available from: 2004-09-16 Created: 2004-09-16Bibliographically approved
List of papers
1. Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles
Open this publication in new window or tab >>Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles
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2001 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1512, no 1, 77-89 p.Article in journal (Refereed) Published
Abstract [en]

Certain short peptides, which are able to translocate across cell membranes with a low lytic activity, can be useful as carriers (vectors) for hydrophilic molecules. We have studied three such cell penetrating peptides: pAntp (‘penetratin’), pIsl and transportan. pAntp and pIsl originate from the third helix of homeodomain proteins (Antennapedia and Isl-1, respectively). Transportan is a synthetic chimera (galanin and mastoparan). The peptides in the presence of various phospholipid vesicles (neutral and charged) and SDS micelles have been characterized by spectroscopic methods (fluorescence, EPR and CD). The dynamics of pAntp were monitored using an N-terminal spin label. In aqueous solution, the CD spectra of the three peptides show secondary structures dominated by random coil. With phospholipid vesicles, neutral as well as negatively charged, transportan gives up to 60% α-helix. pAntp and pIsl bind significantly only to negatively charged vesicles with an induction of around 60% β-sheet-like secondary structure. With all three peptides, SDS micelles stabilize a high degree of α-helical structure. We conclude that the exact nature of any secondary structure induced by the membrane model systems is not directly correlated with the common transport property of these translocating peptides.

Keyword
homeo-peptide, penetratin, transportan, phospholipid vesicle, interaction, secondary structure
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-23347 (URN)10.1016/S0005-2736(01)00304-2 (DOI)000168735500008 ()
Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2017-12-13Bibliographically approved
2. Cellular internalization of a cargo complex with a novel peptide derived from the third helix of the islet-1 homeodomain: Comparison with the penetratin peptide
Open this publication in new window or tab >>Cellular internalization of a cargo complex with a novel peptide derived from the third helix of the islet-1 homeodomain: Comparison with the penetratin peptide
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2001 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 12, no 6, 911-916 p.Article in journal (Refereed) Published
Abstract [en]

Cellular translocation into a human Bowes melanoma cell line was investigated and compared for penetratin and pIsl, two peptides that correspond to the third helices of the related homeodomains, from the Antennapedia transcription factor of Drosophila and the rat insulin-1 gene enhancer protein, respectively. Both biotinylated peptides internalized into the cells with similar efficacy, yielding an analogous intracellular distribution. When a large cargo protein, 63 kDa avidin, was coupled to either peptide, efficient cellular uptake for both the peptide−protein complexes was observed. The interactions between each peptide and SDS micelles were studied by fluorescence spectroscopy and acrylamide quenching of the intrinsic tryptophan (Trp) fluorescence. Both peptides interacted strongly and almost identically with the membrane mimicking environment. Compared to penetratin, the new transport peptide pIsl has only one Trp residue, which simplifies the interpretation of the fluorescence spectra and in addition has a native Cys residue, which may be used for alternative coupling reactions of cargoes of different character.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23348 (URN)10.1021/bc0100298 (DOI)
Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2017-12-13Bibliographically approved
3. Conformational states of the cell-penetrating peptide penetratin when interacting with phospholipids vesicles: effects of surface charge and peptide concentration
Open this publication in new window or tab >>Conformational states of the cell-penetrating peptide penetratin when interacting with phospholipids vesicles: effects of surface charge and peptide concentration
2002 In: Biochimica et biophysica acta Biomembranes, ISSN 0005-2736, Vol. 1563, no 1-2, 53-63 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23349 (URN)
Note
Part of urn:nbn:se:su:diva-247Available from: 2004-09-16 Created: 2004-09-16Bibliographically approved
4. Comparison of the interaction, positioning, structure induction and membrane Perturbation of cell-penetrating peptides and non-translocating variants with phospholipid vesicles
Open this publication in new window or tab >>Comparison of the interaction, positioning, structure induction and membrane Perturbation of cell-penetrating peptides and non-translocating variants with phospholipid vesicles
2003 In: Biophysical chemistry, ISSN 0301-4622, Vol. 103, no 3, 271-288 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23350 (URN)
Note
Part of urn:nbn:se:su:diva-247Available from: 2004-09-16 Created: 2004-09-16Bibliographically approved
5. Cell membrane translocation of the N-terminal (1-28) part of the prion protein
Open this publication in new window or tab >>Cell membrane translocation of the N-terminal (1-28) part of the prion protein
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2002 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 299, no 1, 85-90 p.Article in journal (Refereed) Published
Abstract [en]

The N-terminal (1-28) part of the mouse prion protein (PrP) is a cell penetrating peptide, capable of transporting large hydrophilic cargoes through a cell membrane. Confocal fluorescence microscopy shows that it transports the protein avidin (67 kDa) into several cell lines. The (1-28) peptide has a strong tendency for aggregation and P-structure formation, particularly in interaction with negatively charged phospholipid membranes. The findings have implications for how prion proteins with uncleaved signal peptides in the N-termini may enter into cells, which is important for infection. The secondary structure conversion into beta-structure may be relevant as a seed for the conversion into the scrapie (PrPSc) form of the protein and its arnyloidic transformation.

Keyword
prion protein N-terminus, cell penetrating peptide, aggregation, beta-structure, membrane interaction
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-23351 (URN)10.1016/S0006-291X(02)02595-0 (DOI)000179496700013 ()
Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2017-12-13Bibliographically approved
6. Cell-penetration and membrane leakage by peptides derived from the N-termini of prion proteins
Open this publication in new window or tab >>Cell-penetration and membrane leakage by peptides derived from the N-termini of prion proteins
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(English)Manuscript (Other academic)
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23352 (URN)
Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2016-01-29Bibliographically approved
7. Fluorescence correlation spectroscopy studies on membrane perturbation by peptides derived from the N-termini of prion proteins
Open this publication in new window or tab >>Fluorescence correlation spectroscopy studies on membrane perturbation by peptides derived from the N-termini of prion proteins
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23353 (URN)
Note
Part of urn:nbn:se:su:diva-247Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2010-01-13Bibliographically approved

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