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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
Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. University of Tartu, Estonia.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. Estonian Biocenter, Estonia.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2001. Vol. 12, no 6, 911-916 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-23348DOI: 10.1021/bc0100298OAI: oai:DiVA.org:su-23348DiVA: diva2:191476
Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Cell-penetrating peptides in model membrane systems: Interaction, structure induction and membrane effects
Open this publication in new window or tab >>Cell-penetrating peptides in model membrane systems: Interaction, structure induction and membrane effects
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:nbn:se:su:diva-247 (URN)91-7265-956-4 (ISBN)
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
2. Cell-penetrating peptides and bioactive cargoes: Strategies and mechanisms
Open this publication in new window or tab >>Cell-penetrating peptides and bioactive cargoes: Strategies and mechanisms
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell membrane is an impermeable barrier for most macromolecules. Recently discovered cell-penetrating peptides (CPPs) have gained lot of attention because they can cross the membrane, and even more, carry cargoes with them. How CPPs enter cells is still not clear, while the delivery of different cargoes has been convincingly shown. This thesis concentrates on evaluating CPPs as vectors for different biologically relevant cargoes. Proposed internalisation mechanisms are reviewed as well as cargo coupling strategies. Biological activities of antisense oligonucleotides delivered by CPPs have been of particular interest and are explained in greater details.

A new CPP, pIsl, was derived from Islet-1 transcription factor, and compared to archetypical CPPs like penetratin and transportan. All three peptides resided in the headgroup region of lipid bilayers in model membranes. However, penetratin and pIsl did only interact with negatively charged membranes, while transportan did not distinguish negatively charged and neutral membranes. This suggests different translocation pathways for different CPPs. Biotinylated pIsl and penetratin were complexed with avidin, and uptake of avidin into the human melanoma cell line Bowes was observed in both cases. This means that the protein is not unfolded during the translocation process, which is important in delivery of other, biologically active proteins.

Transportan and its analogue TP10 were used for peptide nucleic acid (PNA) antisense oligonucleotide delivery. First, eight human galanin receptor type 1 targeting PNA oligomers were designed, conjugated to transportan and assayed for antisense efficiency. In contrary to avidin-biotinylated peptide conjugate, a covalent bond between PNA oligomers and the transport peptide was necessary for cellular uptake of oligomers. A common problem in antisense technology is inactivity of antisense oligonucleotides due to the secondary structure of the target. Efficiencies of tested galanin receptor type 1 targeting PNA oligomers varied over two orders of magnitude. The most efficient oligomers were targeting coding sequence regions 24-38 and 27-38, and had EC50 values 70 and 80 nM, respectively.

TP10-antisense PNA oligomer conjugates were targeted also to L-type voltage dependent Ca2+ channel subunits CaV1.2 and CaV1.3. Specific down-regulation of respective proteins was demonstrated by immunohistochemistry. Physiological response to the down-regulation of either of Ca2+ channels was studied by alteration of flexor reflex sensitisation. Rats treated with either of the antisense PNA, but not with scrambled PNA lost the action potential windup phenomenon. In conjunction with a variety of drugs, modulating the conductivity and excitability of neuronal membranes, a central role of L-type CaV channels in sensitisation was confirmed. Nevertheless, also N-methyl-D-aspartate and glycine receptors were found to be required.

Finally, delivery of plasmids by TP10 was evaluated. In contrary to many similar CPPs, TP10 was incapable to translocate plasmids to cells. However, addition of TP10 or a TP10-PNA conjugate to polyethyleneimine-condensed plasmids increased the expression of reporter genes.

In summary, different types of cargoes have been delivered by CPPs and different cargo coupling strategies have been used. CPP-mediated antisense oligonucleotide delivery has been used to identify accessible sites in human galanin receptor type 1 mRNA and to determine the role of L-type voltage dependent Ca2+ channels in axon potential windup.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2004. 75 p.
Keyword
Cell-penetrating peptide, delivery, Peptide nucleic acid
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-308 (URN)91-7265-986-6 (ISBN)
Public defence
2004-12-17, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2004-11-25 Created: 2004-11-25 Last updated: 2017-12-01Bibliographically approved

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Eriksson, L. E. GöranLangel, ÜloGräslund, Astrid
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