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Interactions of Cell-Penetrating Peptides with Membranes 179
Receptor binding of these conjugates was determined at the human neuroblastoma
cell line SK-N-MC, which selectively expresses the NPY Y-1 receptor subtype; cytotoxic
activity was evaluated using c cellular cytotoxicity assay. The different conjugates
were able to bind to the receptor with affinities ranging from 25 to 51 nM, but only the
compound containing the acid-sensitive bond showed cytotoxic activity comparable to
the free daunorubicin and this cytotoxicity was Y-1 receptor-mediated. The intracellular
distribution showed that the active conjugate releases daunorubicin, which is localized
close to the nucleus, whereas the inactive conjugate is distributed distantly from the
nucleus and does not seem to release the drug within the cell.
8.6 UNFOLDING FOR CONJUGATE CPP–PROTEIN
Some transduction domains of proteins such as Tat, Antennapedia, or VP22 appear
to have the highest level of demonstrated protein-transduction efficiency. However,
in some cases the transduction of full-length fusion proteins across the cell membrane
results in an inactivation or denaturation of the protein. This suggested that transduction
across the cellular membrane is a stringent process that partially or completely
unfolds a protein. 20,21
In fact, this is a common aspect for proteins that must penetrate into a membrane
medium. Such a situation can be illustrated by the two following examples in which
membrane-induced conformational changes were detected. The first concerns colicin
A, which adopts the molten globule form to insert the membrane; the membraneassociated
form differs from the hydrosoluble one only by the three-dimensional
organization and not by the secondary structure. 115,116 In the second example, the
toxin cry1Aa of Bacillus thuringiensis, the membrane uptake induces drastic conformational
changes involving a loss of sheet structures. 96 Are these conformational
changes reversible when the proteins are released into the aqueous medium? Almost
nothing is known about this point, but it is likely that recovery of the native hydrosoluble
form will be sequence- and medium-dependent.
8.7 ORIGIN OF THE TOXICITY
Some cell-penetrating peptides show strong toxic properties and, in order to understand
the mechanism underlying these properties, several tests can be carried out.
One consists in measurement of bacteriocidal activity for living organisms such as
bacteria. Among them, mollicutes can be chosen because they represent the simplest,
most cultivable, and most self-replicating organisms described thus far. Their cell
envelope is composed only of the plasma membrane, which should facilitate analysis
of the interactions between peptides and the bacterial cell surface.
The two series of peptides tested in a comparative manner revealed that the
SP–NLS peptides (Table 8.1) were more active than those of the FP–NLS (Table 8.2)
series. This was analyzed based on their effect on spiroplasma motility, cell morphology,
membrane potential, and transmembrane ∆pH. 117
All these experiments suggested that formation of transmembrane ion channel
could be induced by carrier peptides, especially by those of the SP–NLS series.