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116 Cell-Penetrating Peptides: Processes and Applications
along with plasmid transfection used to introduce DNA fragments into cells, 6,7 also
require complex sample manipulation, experienced technical staff, and purchase of
single-purpose systems; the combination of these issues results in delivery techniques
that are inaccessible to many research groups.
To allow simplified noninvasive delivery of peptides, proteins and oligonucleotides
into intact cells a new method termed the “Trojan horse” approach has been
developed. For this, the biologically active cargo is associated with a peptide intrinsically
capable of penetrating the cell membrane lipid bilayer, resulting in delivery
of the cargo to the intracellular space without lethal disruption of the membrane.
Several peptides have been identified that can freely translocate across cell membranes
and reside within the cytoplasm and nucleus without compromising normal
cellular function. The majority of translocating peptides routinely used today have
undergone sequence modifications such as amino acid substitution and formation of
chimeras to produce variants with increased translocation activity compared to the
original native sequences.
In the literature, there are two major classes of translocating peptides. The first
category is highly cationic with arginine- or lysine-rich sequences. They include
HIV-1 Tat peptide, 8 penetratin, 9 the chimeric transportan, 10 and heat shock protein
Hsp70. 11 The second category is hydrophobic and based on protein signal
sequences. 12 These peptides differ in size, composition, and, possibly, mechanism
of entry; yet all exhibit translocation activity. The herpes simplex virus type 1 VP22
protein has also been reported to possess translocation activity that is not dependent
on classical endocytosis or energy. 13 However, Lundberg and Johansson recently
reported findings that suggest that the observed uptake is a product of artificial import
and nuclear localization of VP22 during fixation. 14 O’Hare and Elliott agreed that
some uptake during fixation does occur, but that this is a weak effect and insufficient
to account for total transport activity. 15
This chapter focuses on the identification and use of hydrophobic signal
sequence-based peptides as translocation peptides; detailed descriptions of the
remaining peptides may be found in other chapters of this handbook. Several aspects
critical to the design of effective translocation peptides are also addressed, including
the use of ligation strategies for coupling translocation peptides to cargoes and the
identification of a potentially new class of translocation peptides.
6.2 SIGNAL HYPOTHESIS
Membrane translocating sequence (MTS) peptides are predominantly hydrophobic
in character and therefore unlike the majority of translocating peptides also described
in this book. These peptides are derived from secretory proteins that translocate
through cellular membranes following synthesis and post-translational modifications.
More than 20 years ago, Blobel and co-workers succinctly reported on the
presence of a signal sequence and on the reliance of protein translocation on the
interaction of this signal sequence with the membrane directly or the translocation
machinery. 16-18
Our understanding of the process by which a protein achieves its topological
distribution within a cell or within a membrane has been shaped by Blobel’s work.