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Interactions of Cell-Penetrating Peptides with Membranes 177
that showed that the monolayer uptake of these two peptides was governed by the
hydrophobic domains. 48 This observation raised the following question: since both
peptides bear the same hydrophilic sequence, how can the lipid headgroup-dependent
uptake be governed by the hydrophobic domain?
Some recent preliminary data suggest that the conformational state of this latter
domain could be a determinant factor governing the membrane penetration of the
shuttle peptides. Indeed, starting from the FP–NLS sequence, which is nonordered
in water, several minor changes in the hydrophobic sequence lead to an α-helical
peptide (peptide 6 of Table 8.2) that can penetrate indifferently into neutral and
negatively charged model membranes.
8.3 CELLULAR INTERNALIZATION OF ANTENNAPEDIA
This well-known shuttle peptide based on the third helix of Antennapedia, (R-Q-I-K-
I-W-F-Q-N-R-R-M-K-W-K-K) can also reach the cytoplasm and is eventually conveyed
to the nucleus of cells in culture through a pathway that does not involve classical
receptor-mediated endocytosis. Several observations, such as that the peptide is not able
to induce channel formation, together with the consequences of replacing the two
tryptophan residues by phenylalanines that did not modify the helical structure but
abolished translocation, led to proposal of a model implying inverted micelles
(Figure 8.7). The main reasons for favoring this model are the limited size of the
polypeptidic cargo and its conformation, the requirement of Trp residues, multimer
formation, and formation of hexagonal phases or inverted micelles. 14,114
8.4 INTERNALIZATION VIA A VESICULAR PROCESS
Vesicular internalization is suggested to be associated with self-assembly induced
perturbation of the lipid bilayer. This can be illustrated by study of the mechanisms
involved in translocation of human calcitonin (hCT) through excised bovine nasal
mucosa. 23 Using two carboxyfluorescein-labeled hCT fragments, it was shown that,
in presence of the endocytosis inhibitor cytochalasin D, mucosal-to-serosal and
serosal-to-mucosal hCT permeabilities were equal. Pathway visualization by confocal
laser scanning microscopy showed punctated fluorescence indicating vesicular
internalization. In contrast, the N-terminal fragment lacking the beta-sheet forming
C terminus was not internalized. Circular dichroism showed that, when interacting
with neutral and negatively charged liposomes, hCT adopts beta-sheet conformation.
In a concentrated aqueous solution, beta-sheet formation induces hCT self-assembly
and fibrillation. It was proposed that the high lipid partitioning and beta-sheet
formation result in C terminus-restricted supramolecular self-assembly in lipid membranes.
Condensed hCT self-assemblies may explain the high capacity of net
mucosal-to-serosal hCT permeation, which compares favorably with the low transport
capacity of receptor-mediated endocytosis.
8.5 INTERNALIZATION VIA RECEPTOR
One of the major problems in cancer chemotherapy is severe side effects that limit
the dose of anticancer drugs because of their unselectivity for tumor vs. normal