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Naturally derived cell-penetrating peptides and applications in gene regulation: A study on internalization mechanisms and endosomal escape
Stockholm University, Faculty of Science, Department of Neurochemistry.
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 [en]
peptide, oligonucleotide, endocytosis, gene regulation
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:su:diva-1328OAI: oai:DiVA.org:su-1328DiVA: diva2:189894
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
List of papers
1. Uptake mechanisms of novel cell-penetrating peptides derived from the Alzheimer’s disease associated gamma-secretase complex
Open this publication in new window or tab >>Uptake mechanisms of novel cell-penetrating peptides derived from the Alzheimer’s disease associated gamma-secretase complex
2006 (English)In: International journal of peptide research and therapeutics, ISSN 1573-3149, Vol. 12, no 2, 105-114 p.Article in journal (Refereed) Published
Abstract [en]

Basic peptides with vector abilities, so called cell-penetrating peptides (CPPs), have been reported to enter cells, carrying cargoes ranging from oligonucleotides and proteins to nanoparticles. In this study we present novel CPPs derived from the gamma-secretase complex, which is involved in the amyloidogenic processing of the amyloid precursor protein (APP) and one of the major research targets for Alzheimer’s disease therapeutics today. In order to examine the uptake efficiency and internalization mechanism of these novel CPPs, side-by-side comparison with the well characterized CPPs penetratin and tat were made. For assessment of the CPP uptake mechanism, endocytosis inhibitors, endosomal markers and cells deficient in the expression of glycosaminoglycans were used. Also, in order to determine the vector ability of the peptides, protein delivery was quantified.

We demonstrate the uptake of the gamma-secretase derived CPPs, in accordance to penetratin and tat, to be largely dependent on temperature and initial binding to cell-surface glycosaminoglycans. After this initial step, there is a discrepancy in the mechanism of uptake, where all peptides, except one, is taken up by a PI 3-kinase dependent fluid phase endocytosis, which could be inhibited by wortmannin. Also, by using endosomal markers and protein delivery efficacy, we conclude that the pathway of internalization for different CPPs could determine the possible cargo size for which they can be used as a vector. The, in this study demonstrated, cell-penetrating properties of the gamma-secretase constituents could prove to be of importance for the gamma-secretase function, which is a matter of further investigation.

Keyword
Cell-penetrating peptide, CPP, internalization, endocytosis, Alzheimer’s disease, gammasecretase
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23005 (URN)10.1007/s10989-005-9007-y (DOI)
Available from: 2006-10-31 Created: 2006-10-31 Last updated: 2015-04-21Bibliographically approved
2. N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis
Open this publication in new window or tab >>N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis
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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.

Keyword
Prion protein, N-terminus, Cell-penetrating peptide, Endocytosis, Macropinocytosis, Proteoglycan
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23006 (URN)10.1016/j.bbrc.2006.07.065 (DOI)
Available from: 2006-10-31 Created: 2006-10-31 Last updated: 2015-04-21Bibliographically approved
3. Studying the uptake of cell-penetrating peptides
Open this publication in new window or tab >>Studying the uptake of cell-penetrating peptides
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2006 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 1, no 2, 1001-1005 p.Article in journal (Refereed) Published
Abstract [en]

More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-20766 (URN)10.1038/nprot.2006.174 (DOI)17406337 (PubMedID)
Available from: 2008-01-14 Created: 2008-01-14 Last updated: 2015-04-21Bibliographically approved
4. Induction of splice correction by cell-penetrating peptide nucleic acids
Open this publication in new window or tab >>Induction of splice correction by cell-penetrating peptide nucleic acids
2006 (English)In: Journal of Gene Medicine, ISSN 1099-498X, E-ISSN 1521-2254, Vol. 8, no 10, 1262-1273 p.Article in journal (Refereed) Published
Abstract [en]

Background

Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as β-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing.

Methods

HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 °C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs.

Results

All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 °C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis.

Conclusions

These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.

Keyword
antisense, cell-penetrating peptide, endocytosis, PNA, splice correction
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23008 (URN)10.1002/jgm.950 (DOI)
Available from: 2006-10-31 Created: 2006-10-31 Last updated: 2015-04-21Bibliographically approved
5. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides
Open this publication in new window or tab >>Delivery of short interfering RNA using endosomolytic cell-penetrating peptides
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2007 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 21, no 11, 2664-2671 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.—Lundberg, P., El-Andaloussi, S., Sütlü, T., Johansson, H., Langel, Ü. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides.

Keyword
CPP, gene silencing, siRNA, oligonucleotides, RNA interference
National Category
Neurosciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23009 (URN)10.1096/fj.06-6502com (DOI)
Available from: 2006-10-31 Created: 2006-10-31 Last updated: 2015-04-21Bibliographically approved

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