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100 Cell-Penetrating Peptides: Processes and Applications
Amphipathic sequences contain a periodicity of hydrophobic and polar residues,
such as the fusion peptide of influenza hemaglutinin (HA-2), 53-55 and related synthetic
analogs called GALA, KALA, 56,57 JTS1, 58 and Hel 11.7. 59 Import signal peptides
have been demonstrated to interact with cellular membranes and lipid bilayers,
resulting in fusion events with the membrane. The conformation of these peptides
is sensitive to the pH: acidic pH induces a conformational change from random coil
to a helical transition that induces leakage of vesicular contents. They have been
used in association with an NLS in order to improve release from the endosomes
of different formulations. 53
Another class of cell-penetrating peptides corresponds to the protein transduction
domains and includes the third helix of Antennapedia, 53 Tat HIV protein, 2 VP22
protein, 61,62 and transportan. 63 These peptides have been proposed to enter the cell
through a mechanism independent of the endosomal pathway and will be described
in detail in other chapters of this book.
5.3.3 APPLICATIONS FOR NLS IN CELL-PENETRATING PEPTIDES
NLS peptides covalently linked to a variety of proteins have already been demonstrated
to increase the potential for nuclear localization and to confer nuclear localization
to nonnuclear proteins. Moreover, NLSs have been associated with different
hydrophobic cell-penetrating peptides in order to favor membrane crossing of nuclear
targeting cargoes. Finally, NLSs have been associated with cationic peptides or
motifs to facilitate DNA binding and compaction for gene delivery. 8-10
Several bifunctional peptides containing NLS have been described (Table 5.1).
Braden et al. have proposed a bifunctional peptide (PNA–NLS) combining a peptide
nucleic acid (PNA) with the core nuclear localization sequence of SV40 for gene
delivery. PNAs are synthetic molecules which bind DNA with high affinity and
specificity. 64,65 They have been used to label specific locations of DNA with high
interactive specificity and stability. 66 PNA–NLS associated with polyethyleimide
dramatically increase the nuclear uptake of oligonucleotides and facilitate plasmid
transfection in an NLS-dependent fashion. PNA–NLS-conjugated molecules constitute
a promising tool for therapeutic gene therapy and have recently been used
successfully for in vivo nuclear delivery of ODN. 67 However, one of the limitations
of this approach resides in the choice of the PNA target site to promote efficient
transfection.
The NLS of NFkB p50 subunit was coupled with the cell membrane-permeable
motif carrier derived from the hydrophobic region of the signal peptide of Kaposi
fibroblast growth factor (K-FGF). This cell penetrating peptide was shown to block
intracellular recognition of the NLS and to inhibit nuclear translocation of NF-κB
transcription factor. The authors demonstrated that the biological effects were due
to the NLS, as transport was energy dependent and completely abolished upon
mutation or deletion of the NLS. 68
Several studies have shown that cross-linking an NLS to polycationic peptides
(pLys) can enhance transfection by specifically conferring recognition by nuclear
import receptors. Peptide P101 associated with polylysine improves recognition by