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14 Cell-Penetrating Peptides: Processes and Applications
the short HIV-1 Tat peptide did not enter cells via this receptor type (Silhol et al.,
in press). Briefly, we made use of various CHO mutant cell lines totally or partially
deficient in the expression of heparan sulfate proteoglycans 44 and clearly established
that rhodamin- or fluorescein-labeled Tat CPP was normally taken up in these mutant
cell lines. The internalization of the Tat peptide in these cell lines was monitored in
parallel by fluorescence microscopy and by FACS scan analysis. In addition, the
digestion of HS receptors by heparinase III prior to incubation with the Tat entities
abolished the internalization of the GFP-fused Tat protein, but did not alter the uptake
of the Tat CPP (Silhol et al., in press).
Tat CPP cellular entry does not seem to involve the endocytosis pathway since
the incubation of cells with Tat fluorochrome-labeled peptides never induced the
characteristic punctuated cellular labeling of endocytic vesicles. 23 This is not definitive
proof, however, since passage through the endocytic compartment could be
very transient as observed with cationic lipids delivery. On the other hand, it was
recently reported that sodium azide, which inhibits the cellular oxidative phosphorylation
process, prevented Tat peptide analogues uptake, 47 in keeping with an
energy-dependant pathway.
In contradiction to these latter data, low temperature (4°C) incubation of cells
with short Tat peptides containing the full cluster of basic amino acids did not abolish
internalization. 23 Internalization of the Tat CPP uptake at low temperature also took
place when the short Tat peptide was chemically bound to liposomes 42 or was
complexed to a chelating moiety. 48 Although internalization of the Tat CPP takes
place at low temperature, a quantitative evaluation performed by FACS analysis
indicated a decrease of the level of Tat CPP internalization during incubation at low
temperature (Silhol et al., in press). Likewise, a 60% reduction of the internalization
rate of the Antennapedia CPP has been recently demonstrated by FACS analysis. 49
Here again, fluorescence microscopy analysis of the Antennapedia CPP distribution
had not revealed a diminished uptake at low temperature. 17
Whether slightly reduced CPP uptake at low temperature is really significant
must be further analyzed. Along these lines, another CPP peptide called Transportan,
designed from the fusion of a peptide derived from the neuropeptide galanin and
the mastoparan, was also shown to translocate efficiently through the plasma membrane
at 4 and 0°C. 50 Despite these apparently contradictory results between sodium
azide treatment and low temperature incubation, data indicate that endocytosis is
not the major pathway allowing the cellular uptake of the Tat peptide.
1.4.3 MOLECULAR ASPECTS OF TAT CPP UPTAKE
Internalization of these CPPs in various cell lines did not involve the presence of a
specific cellular receptor or transporter, as demonstrated for the Antennapedia
peptide 17 and discussed in another section in this book. The same conclusion could
also be made for the Tat CPP since several analogues derived from the Tat peptide
retained their ability to be internalized in cells. The more convincing results, which
excluded a receptor interaction, have been provided by the use of a Tat CPP assembled
with d-amino acid derivatives (inverso) and from a Tat CPP synthesized from
the C-terminal end to the N-terminal end (usually called retro sequence). 47,51 The