crc press - E-Lib FK UWKS

Hydrophobic Membrane Translocating Sequence Peptides 129
Naturally occurring nuclear localization signal (NLS) sequences responsible for
the import of proteins into the nucleus have also been exploited as translocation
peptides. These peptides are also cationic in nature and capable of traversing cell
and nuclear membranes. 132-134 There are two types of NLS peptides: monopartite,
which consist of a single cluster of basic residues, or bipartite, which contain two
clusters of basic residues. 135-137 Examples of monopartite and bipartite NLS
sequences are shown in Table 6.2.
According to the established model of nuclear translocation, the NLS sequence
on the protein surface is bound by an import receptor complex located in the
cytoplasmic side of the nuclear membrane. The protein is then directed to a nuclear
pore 138-140 where a cascade of tightly regulated interactions results in delivery of the
protein into the nucleus. The protein is subsequently released from the importin
complex. Furthermore, because this is a unidirectional event, it is a highly efficient
means for delivering biologically active molecules into the nucleus. The definition
of an NLS sequence is vague because of the diversity of peptides that can apparently
function in this manner. 136 However, a quantitative method developed by Hodel
et al. 141 can distinguish between efficient and weak NLS peptides on the basis of
their affinity for the importin complex.
6.7 ARGININE–PROLINE-RICH PEPTIDES
AS TRANSLOCATING PEPTIDES
Peptides with a high content of arginine and proline are heavily represented in
antimicrobial peptides, a particular class of peptides produced by a range of species;
furthermore, these peptides translocate across the cell membrane. 142-144 Antimicrobial
peptides are classified on the basis of their structure and peptide composition: linear
peptides that form α-helices; cyclic peptides rich in disulphide bonds; and peptides
with a high content of a particular amino acid, usually proline or glycine. 145 Examples
of the arginine–proline-rich (RP-rich) peptides include PR-39, 146,147 abaecin, 148 apidaecin,
149 Bac5, and Bac7. 150,151 The bactericidal properties of amphipathic, α-helical
peptides (cecropins, magainins) and the disulphide-containing peptides (defensins)
are thought to relate to their ability to form a pore in the target membrane, 152,153
whereas the RP-rich peptides penetrate the membrane and subsequently kill cells
by interfering with the protein synthesis machinery. 142,154,155
The antimicrobial activity exhibited by RP-rich Bac7 is retained by the 24-residue
peptide RRIRPRPPRLPRPRPRPLPFPRPG 156 that represents the N terminus. 157
To determine if this peptide and a series of truncated peptides could traverse the cell
membrane each peptide was synthesized with a fluorophore attached to the
N terminus and incubated with murine monocytes. It was found that peptides with
both a high and low content of arginine could translocate into cells, dispelling the
hypothesis that a high content of arginine was solely responsible for the translocation.
158 The maximum intracellular concentration of fluoresceinated peptides
occurred within 5 min and then decreased at a steady rate, indicating that rapid
translocation occurs. Preliminary cytotoxicity assays indicated that the fluoresceinated
peptides are not cytotoxic at concentrations up to 100 µM after exposure for 24 h.