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Signal Sequence-Based Cell-Penetrating Peptides 99
5.3 NLS-CONTAINING CELL-PENETRATING PEPTIDES
5.3.1 SIGNAL SEQUENCE-BASED PEPTIDES: IMPORT AND TARGETING SIGNALS
Bifunctional peptide sequences containing NLS have been designed to overcome
both cytoplasmic and nuclear entry barriers. The poor permeability of the plasma
membrane of eukaryotic cells to drugs or DNA represents the first limiting barrier
to the development of therapeutic molecules. A series of peptides able to cross the
plasma membrane have been described and will be developed in detail in other
chapters of this book. Here we will essentially focus on chimeric cell-penetrating
peptides in which a nuclear targeting sequence has been coupled to an import peptide
sequence. 5,7,9
In general, cell-penetrating peptide-based import signals must be capable of
directing the movement of a cargo across the cell membrane into the cytoplasm of
most of the cells, either by transient membrane permeabilization or through the
endocytotic pathway by receptor-mediated endocytosis. In order to facilitate the
rapid internalization of NLS-containing peptides, import signal sequences selective
to specific cells and receptors have been used. 9,10 However, the delivery of chemotherapeutic
drugs imported through this mechanism appears to be limited to certain
cell lines. To address this issue, several short import signal sequence peptides have
been coupled to NLSs and can be divided into three major classes: hydrophobic,
amphiphatic, or cationic peptides. 4
5.3.2 IMPORT SIGNAL SEQUENCES
The first class of import signal sequences corresponds to the hydrophobic region of
various signal sequences termed membrane-permeable sequences (MPSs). 10,44 MPSs
are about 20 amino acids long and adopt a characteristic helical conformation under
membrane mimetic environment, irrespective of their distinct primary sequences.
They are capable of crossing the cell membrane and importing covalently attached
functional domains from other intracellular proteins. A series of peptides have been
described so far, including the Src homology 2 (SH2) domain of Grb2, 45 human
integrins β1, β3, 46 and the sequence signal of Kaposi fibroblast growth factor (K-
FGF) AAVAALLPAVLLALLAP. 8,10 The mechanism of internalization of these peptides
does not require receptor proteins and is independent of the classical endocytotic
pathway. 10 The alpha-helical structure of these peptides seems to be required
for penetration but is not sufficient by itself for cellular delivery. Studies have
demonstrated that the versatility of the structure of the hydrophobic domain is
important for cell penetration.
A series of peptide fusion sequences and signal sequences have also been used,
including the hydrophobic signal sequence from the Caiiman crocodylus immunoglobulin
light chain signal sequence and fusion sequence from HIV gp41 fusion
peptide. 47-52 Peptides such as IgV (light) chain of caiman crocodylus associated with
an NLS and helical h-FGF were shown to adopt a beta strand fold. 47 Such sequences
have been fused to the NLS of SV40 large T antigen to target the nucleus of cells
and to deliver antisense oligonucleotides, peptides, plasmid DNA, and drugs. 48-51