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224 Cell-Penetrating Peptides: Processes and Applications<br />

10.10 Speculations on the Mechanisms of the CPP Translocation ....................240<br />

Acknowledgment ...................................................................................................241<br />

References..............................................................................................................241<br />

10.1 INTRODUCTION<br />

Cell-penetrating peptides, CPPs, are water-soluble but have the ability to translocate<br />

through various cell membranes with high efficiency and low lytic activity. The<br />

transport is nonendocytoic, does not require a chiral receptor, and is non-cell-type<br />

specific. 1-3 When covalently linked to “cargos” (polypeptides, oligonucleotides, particles,<br />

etc.) many times their own molecular mass, CPPs still retain their translocating<br />

ability.<br />

Examples of CPPs are so-called Tat-derived peptides, as well as peptides based<br />

on sequences from certain transcription factors, signal sequences, chimeras between<br />

biological sequences, or completely synthetic (artificial) peptides. The most studied<br />

are those with sequences derived from homeodomains, where “penetratin” peptide<br />

is an archetype among the CPPs. Homeodomain (homeobox) proteins belong to a<br />

class of transcription factors involved in multiple morphological processes. 3 The<br />

sequences of penetratin, transportan, and a few other CPPs are shown in Table 10.1.<br />

The mechanisms behind translocation of the CPPs are still mostly unknown,<br />

although it is clear that the molecular details of peptide–membrane interaction are<br />

fundamentally important for the translocation process. The role of any particular<br />

secondary structure in the process, though, remains disputed.<br />

Background for the present review is the overall question of translocation mechanism<br />

of CPPs; however, with the lack of unambiguous experimental evidence<br />

regarding this question, we will mainly focus on membrane interaction properties<br />

of certain CPPs and analogues. Membrane model systems will be presented and the<br />

various biophysical methods used will be briefly described. We will also make a<br />

short comparison with studies on other classes of bioactive, membrane-interacting<br />

peptides in which more is known about the mechanisms of action.<br />

10.2 SEQUENCES AND GENERAL PROPERTIES OF SELECTED CPPS<br />

The sequences of selected CPPs discussed in this review are shown in Table 10.1,<br />

together with some variant sequences reported to have lost their translocating property.<br />

Penetratin, denoted pAntp, has a sequence corresponding to the 16 residues of<br />

the third α-helix (residues 43–58) from the Antennapedia homeodomain protein of<br />

Drosophila. 1,4-6 This third helix was found to be responsible not only for interaction<br />

with DNA by binding specifically to cognate sites in the genome, but also for<br />

translocation of the entire protein across cell membranes. 4,5 The pAntp peptide alone<br />

retains the membrane translocation properties and has therefore been proposed as a<br />

universal intercellular delivery vector. 1 A homologous peptide, pIsl, derived from<br />

the Islet-1 homeodomain, also behaves as an efficient CPP. 7<br />

Transportan is a chimeric 27-amino acid peptide with a sequence derived from<br />

the 1–12 residues of galanin and linked with a Lys to the sequence of the wasp

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