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122 Cell-Penetrating Peptides: Processes and Applications
Based on the success of this protein import study, Zhao et al. 83 attached the same
MTS covalently to antibodies, thus creating proteins capable of translocating into
living fibroblast cells without affecting cell structure and viability. The internalized
antibody appeared uniformly in the cytoplasm, whereas the nucleus showed significantly
less antibody-positive material. Importantly, these imported antibodies can
be detected by ELISA after extraction. More recently, by fusion to the 12-residue
kFGF MTS, the DNA Cre recombinase has also been demonstrated to be cellpermeable
and functional in cells in vitro and in vivo. 84
In a significant step forward in the use of MTS peptides as therapeutic and
diagnostic agents Li et al. 85 showed that the SA peptide containing the h-region of
kFGF and the NLS of FGF-1 34 was capable of in vivo cargo delivery. The peptide
was injected directly into the left lateral cerebroventricle of male Wistar rats to
determine the effect of the presence of the FGF-1 NLS on food intake. Compared
to previous studies in which other peptides encompassing the FGF-1 N terminus
were investigated using a similar method, 86,87 the SA peptide exerted a far stronger
inhibitory effect on food uptake. Because the MTS does not affect feeding behavior, 85
it may be concluded that the increased potency of SA peptide is most likely due to
more effective intracellular delivery of the FGF-1 NLS. Another good example of
the therapeutic application of MTS peptides is the role of an SN50 peptide analog
in the inhibition of acute inflammatory disease models. 78
6.3.4 IDENTIFICATION OF TRANSLOCATION ACTIVITY OF INTEGRIN β 3 h-REGION
The favorable results achieved using the kFGF h-region and modified sequences as
MTS peptides seem to have precluded the investigation of h-regions from other
proteins as translocation peptides. Another sequence examined as a potential cellpermeant
peptide vector is the h-region of human integrin β 3. 88 The hydrophobic
peptide VTVLALGALAGVGVG 89 was synthesized in tandem with a set of four
overlapping peptides representing segments of the cytoplasmic tail of human integrin
β 3. This series of peptides was used to identify the region of the integrin β 3 cytoplasmic
tail is involved in the regulation of fibrinogen binding to the extracellular
domain. By incorporating the translocating peptide into the peptides tested, the
authors successfully identified the major cell adhesion regulatory domain (CARD)
of integrin β 3.
6.4 MECHANISM OF MTS MEMBRANE TRANSLOCATION
While the exact mechanism utilized by MTS peptides to translocate across the
plasma membrane is still not known, several important clues have been uncovered.
First, import is not sequence specific because synthetic peptides containing MTSs
from distinct protein signal sequences or modified sequences can translocate into
the intracellular space. 33,36,88 The translocation is also not limited to a particular cell
type; efficient uptake has been observed with many cell types, e.g., human monocytes,
endothelial cells, T lymphocytes, fibroblasts, murine endothelial cells, and
mast cells. 90 This is in contrast to the cell-specific uptake associated with the 60-residue
antennepedia homeobox domain (from which penetratin is derived), where the