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28 Cell-Penetrating Peptides: Processes and Applications
are crucial for translocation into neuronal cells. Fischer et al. studied the effect of
successive truncations of the 43–58 peptide. 16 C-terminal truncations had a dramatic
negative effect on cellular translocation (43–57 and 43–54 mutants, Table 2.2); the
deletion of only the last Lys residue strongly impaired peptide penetration. In
contrast, N-terminal truncations were less detrimental, allowing the definition of a
heptamere (Arg-Arg-Met-Lys-Trp-Lys-Lys, Table 2.2) internalized with 60% efficiency,
compared to full-length penetratin-1. The latter result, at odds with earlier
reports, 15,17 could be explained by the different types of cells used (immortalized
HaCat fibroblasts and lung cancer A549 cell lines instead of neurons). Another
unexplored possibility is the different spacers (amino-pentanoic acid or β-alanine)
used to separate the peptide from the biotin moiety.
2.3.1.5 Relative Importance of Each Individual Residue
In two separate studies, each amino acid of the penetratin-1 sequence was substituted
by alanine-scanning. 16,18 These two studies differ by cell types (human HaCat and
A549 vs. human transformed leukemia cells K562) and by mode of detection (biotin
and NBD fluorochrome (7-nitrobenz-2-oxa-1, 3-diazol-4-yl)). The two reports confirm
the key role of several basic residues: Lys 58, Lys 57, Lys 55, Arg 53, Arg 52,
and Lys 46. The role of hydrophobic residues is less clear, with a distinct controversy
on the function of Trp48 and Trp56. 15,16,18
Taken together, the results from all groups suggest the involvement of basic residues
and of at least one tryptophan in the translocation process. This proposition has been
confirmed by biophysical experiments demonstrating a role of charged groups in
lipid binding and of one tryptophan in lipid destabilization and peptide translocation.
2.3.2 PEPTIDE–LIPID INTERACTIONS
Translocation of the penetratin peptides does not require chiral receptors and does
not involve classical endocytosis. Fluid phase pinocytosis can be excluded on the
basis of ultrastructural studies that failed to reveal any association of penetratins
with vesicular structures; internalization through inverted micelles was proposed.
This model is based on the induction of inverted micelles by tryptophan residues, 20
and on the capacity of penetratins to form multimer in the presence of SDS and to
lower the critical micellar concentration in the presence of lipids. 15,21
To evaluate the implication of inverted micelles in peptide translocation, in vitro
peptide and lipid interactions were analyzed by several laboratories. These experiments
were based on fluorescence spectroscopy, circular dichroism (CD), and
nuclear magnetic resonance (NMR) of proton ( 1 H-NMR) and phosphorus ( 31 P-
NMR). The first series of experiments with 31 P-NMR demonstrated that the addition
of penetratin peptides to lipids from embryonic rat brains induces formation of
inverted micelles. 15,21 In contrast, the double Phe non-internalized variant failed to
induce inverted micelle formation (unpublished results). Furthermore, 1 H-NMR
spectroscopy demonstrated that the conformational flexibility of the peptide backbones
allows their adaptation to the concave surface of an SDS micelle and the
convex surface of an inverted micelle. 21