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crc press - E-Lib FK UWKS

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Hydrophobic Membrane Translocating Sequence Peptides 123<br />

ex<strong>press</strong>ion of α-2,8-polysialic acid on cell surface neuronal cell adhesion molecules<br />

(NCAM) is required. 91 Uptake by T lymphocytes indicates that the mechanism<br />

involved does not depend on membrane caveolae because T lymphocytes do not<br />

ex<strong>press</strong> these structures. 37 The addition of inhibitors of the endosomal pathway to<br />

cell culture does not prevent the intracellular localization of MTS peptides; therefore,<br />

uptake does not rely on endocytosis. 33<br />

Furthermore, in a study using electron microscopy co-staining techniques,<br />

MTS-cargo peptides have been observed to translocate across the cell membrane by<br />

free penetration and without specific association with any membrane-bound vesicles,<br />

such as endosomes. 92 These observations, together with results suggesting that<br />

import of MTS peptides is ATP-independent and temperature-dependent, and that<br />

intact architecture of the plasma membrane is required, 33 lead to conclusions that<br />

translocation occurs directly through the lipid bilayer and that membrane fluidity<br />

and lateral mobility of membrane proteins influence this import process.<br />

The secondary conformation adopted by MTS peptides is potentially responsible<br />

for membrane interaction. The residues located in the h-regions of signal sequences<br />

are often those with a propensity to form α-helices; data from circular dichroism<br />

(CD) studies show that various signal peptides can adopt an α-helical conformation<br />

in membrane-mimetic environments. 31,93-98 Similarly, the correlation of the stability<br />

of the helix–turn–helix conformation of h-regions with in vivo function is observed<br />

with the prokaryotic LamB protein. 99 Furthermore, it has been suggested that a<br />

helical hairpin structure makes hydrophobic peptides prone to interaction with membrane<br />

lipid 93-95,100 and that this interaction unloops the peptide, resulting in a transmembrane<br />

conformation. 101<br />

Du and co-workers performed a detailed study aimed at defining the conformational<br />

requirements of cell-permeant hydrophobic MTS peptides and the correlation<br />

between translocation activity and sequence topology. 102 For this, SA peptide with<br />

sequence AAVALLPAVLLALLAPAAANYKKPKL 34 and several variants were synthesized<br />

and analyzed for secondary conformation and translocating activity. The<br />

16 N-terminal residues of SA represent the h-region of kFGF, which is separated<br />

from the NLS of FGF-1 by a trialanine spacer region. Results confirm that the MTS<br />

induces α-helix formation in membrane-mimetic environments and the authors concluded<br />

that a tendency to adopt α-helical conformation in a heterogeneous<br />

amphiphilic environment suggests a possible interaction with hydrophilic headgroups<br />

of plasma membrane lipids.<br />

These findings suggest that signal sequence-based MTS peptides can be modified<br />

to retain or improve their import activity as long as the secondary conformation<br />

remains unchanged. In fact, Rojas et al. have designed a 12-residue MTS derived<br />

from the 16-residue h-region of kFGF mentioned above. They have shown that this<br />

shortened MTS peptide is also capable of efficiently delivering peptides and proteins<br />

into cells. 36 This 12-residue MTS may be the shortest hydrophobic translocating<br />

peptide reported thus far.<br />

Du et al. also addressed the effect of the location of the MTS on peptide import<br />

function. It was found that the peptide still adopted an α-helical structure in the<br />

membrane-mimetic environment and was able to penetrate across the cell membrane<br />

when the MTS was attached at the C terminus of the NLS. 102 Thus, the site of

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