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crc press - E-Lib FK UWKS

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

61 RNTVNRLLPM LRRKKNEKKN EKIERNNKLK QPPPPPNPND PPPPNPNDPP PPNPNDPPPP<br />

121 NPNDPPPPNA NDPPPPNAND PAPPNANDPA PPNANDPAPP NANDPAPPNA NDPAPPNAND<br />

181 PAPPNANDPP PPNPNDPAPP QGNNNPQPQP RPQPQPQPQP QPQPQPQPQP RPQPQPQPGG<br />

241 NNNNKNNNND DSYIPSAEKI LEFVKQIRDS ITEEWSQCNV TCGSGIRVRK RKGSNKKAED<br />

301 LTLEDIDTEI CKMDKCSSIF NIVSNSLGFV ILLVLVFFN<br />

FIGURE 6.3 Amino acid sequence of P. berghei CS protein. 165 The proline-rich repeat region<br />

is shown in bold.<br />

6.8 PROLINE-RICH PEPTIDES AS TRANSLOCATING PEPTIDES<br />

The translocation activity of a potentially new class of translocating peptides, proline-rich<br />

peptides, has been investigated. Even though each translocating peptide<br />

identified thus far has proven capable of traversing the cell membrane and delivering<br />

biologically active cargo into the cytoplasm and nucleus, several disadvantages are<br />

associated with use of these peptides. For example, even though the basic residues<br />

of cationic peptides seem to aid in interaction with the negatively charged lipid<br />

bilayer and increase translocation, such highly charged peptides are potentially<br />

cytotoxic. In a report published by Singh et al., 159 a loligomer comprising eight<br />

copies of a polylysine sequence had a cytotoxic effect on various eukaryotic cells<br />

at concentrations above 5 µM. Linear polymers of polylysine have also been shown<br />

to possess cytotoxic properties. 160<br />

In a preliminary study we have examined the translocation activity of a peptide<br />

derived from the proline-rich repeat region of the Plasmodium berghei circumsporozoite<br />

surface (CS) protein. The CS protein is the major surface protein of the<br />

Plasmodium sporozoite, 161 the causative agent of malaria. Both the CS protein and<br />

the entire sporozoite possess the ability to translocate from the extracellular environment<br />

to the cytoplasm of hepatocytes. 162-164 Sporozoites have even been shown<br />

to pass back through the membrane and exit cells without fatally disrupting the<br />

membrane. 164 This protein contains a large proline-rich repeat region, the sequence<br />

of the P. berghei CS protein 165 is shown in Figure 6.3. The sequence PPPPNPND-<br />

PPPPNPND was selected for examination of translocation activity; uptake of this<br />

peptide by murine monocytes was found to be greater than that of an RP-rich control<br />

peptide derived from Bac7. 158 The successful uptake of proline-rich peptides has led<br />

to ongoing studies in which the mechanism of uptake is elucidated. These prolinerich<br />

peptides have the desirable properties of being aqueous soluble and not highly<br />

charged and, therefore, more conducive to routine synthesis and delivery.<br />

6.9 FUTURE PERSPECTIVES OF CELL-PERMEANT<br />

PEPTIDE TECHNOLOGY<br />

Peptides should possess several critical properties to be considered translocating vectors:<br />

• Sequence short to simplify synthesis<br />

• Internalization not appreciably affected by coupling to cargo<br />

• Cargo delivered to the desired compartment<br />

• Biologically inactive vector

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