crc press - E-Lib FK UWKS

Signal Sequence-Based Cell-Penetrating Peptides 101
the nuclear import machinery and enhances transfection efficiency. 33,34 An interesting
system was described using a recombinant fusion peptide containing a Gal4 DNAbinding
domain associated to a fragment of the invasin protein of Yersinia pseudotuberculosis.
69 The Gal4 moiety of the system confers DNA-binding and nuclear
targeting characteristics through a mechanism independent of the importin β pathway.
However, Gal4 DNA-binding and nuclear targeting properties are mutually
exclusive, leading to inefficient transport of DNA into the nucleus. In order to use
the inherent property of Gal4 to bind DNA and preserve nuclear targeting characteristics,
the Gal4 peptide was coupled with SV40 NLS polylysine. Association of
Gal4 with an NLS has been used to improve its transfection efficiency. 69,70
Bacterial NLSs containing proteins were also used for nuclear import of DNA
into mammalian cells. Agrobacterium virulence proteins VirD2 and VirE2 have been
demonstrated to be efficient in promoting DNA delivery into Hela cells. Nuclear
import is dependent on α importin and Ran GTP protein. 71
Peptide vehicles that integrate multiple targeting and routing signals are often
limited due to synthetic constraints and domain presentation issues associated with
linear assemblies of efficient NLS-conjugated proteins. For example, the simple
cross-linking between an NLS and an MPS does not work in numerous cases, and
insertion of a linker sequence is essential to preserve the properties of these
domains. 72 In order to control these parameters, branched peptides or L-oligomers
have been developed containing cytoplasmic translocating sequence signals and
nuclear localization signals. L-oligomers corresponding to peptides that integrate
multiple targeting and routing signals have been developed involving polylysine and
NLS. These branched peptides have been used as nonviral transfection agents for
the delivery of proteins and genes. 4,73 Branched peptides are rapidly internalized by
endocytosis and can target and accumulate in different subcellular compartments. 4,74
The signal sequence of the Ig(v) light chain of caiman crocodilus or the fusion
peptide of HIV-1 gp41 has been associated to the hydrophilic nuclear localization
sequence of SV40 large T antigen 5 to develop a single chain peptide-vector (FP-
NLS: GALFLGFLGAAGSTMGAWSQPKKKRKV referred to hereafter as MPG or
SP-NLS: MGLGLHLLVLAAALQGAWSQPKKKRK) which efficiently delivers
short oligonucleotides and drugs into a wide variety of cell lines independently of
the endosomal pathway. 48-51 The FP-NLS peptide family interacts strongly with
nucleic acids through its NLS domain and forms peptide–peptide interactions
through the gp41 hydrophobic domain, thus generating a peptide cage around the
plasmid. After crossing the cell membrane, the presence of the NLS domain promotes
rapid delivery of the plasmid into the nucleus.
Structural and mechanistic investigations have revealed that the flexibility
between the two domains of MPG is crucial for gene delivery and can be improved
by adding a linker sequence between the fusion and the NLS motifs. 5,75 MPG was
shown to deliver small hydrophobic peptides using a similar nonendosomal pathway.
Activity of this peptide is associated to a very versatile structure, and greatest activity
is associated with β-strand structure, whereas α-helical folding is associated with
toxicity. MPG activity is independent of the endosomal pathway and its NLS enables
it to deliver and express transgenes in the nucleus of nondividing cells (Figure 5.4).