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Penetratins 37 2.4.3.1.2 Regulation of Signal Transduction Intracellular protein kinase C (PKC) activity was down-regulated in neurons by the penetratin-coupled PKC pseudo-substrate, provoking growth cone collapse. 31 This confirmed PKC involvement in the transduction of extracellular signals regulating axon elongation. Penetratin-coupled inhibitory peptides targeting protein kinase C epsilon (PKC-ε) have highlighted its role in the protection of ischemic rabbit cardiomyocyte against osmotic shock. 58 Another intracellular kinase, the cGMP-dependent protein kinase I (cGPK) was impaired by peptide blockers fused to penetratin. This inhibition into cerebral arteries cultured in vitro revealed a central role of the kinase in promoting NO-induced vasodilatation. 59 The intracytoplasmic interactions between fibroblast growth factor receptor 1 (FGFR1) and SH2 (sarc-homology 2) domain of phospholipase C have been antagonized with a penetratin-coupled phosphopeptide corresponding to the phosphorylated site of the cytoplasmic domain of FGFR1. 60 This inhibited phospholipid hydrolysis stimulated by basic FGF and, in consequence, neurite outgrowth stimulated by basic FGF. Peck and Isacke have analyzed the signaling pathway induced by an extracellular matrix glycosaminoglycan, the hyaluronan, which leads to cell migration. 61 Protein CD44, the hyaluronan transmembrane receptor, plays an essential role in many physiological events, including tumor progression, lymphocyte homing, and tissue morphogenesis. Hyaluronan binding to CD44 induces its phosphorylation on a serine crucial for hyaluronan-dependent cell migration. Penetratin-linked peptides mimicking endogenous phosphorylation sites blocked CD44-induced cell migration without reducing its expression or ability to bind hyaluronan. Penetratin-coupled peptides have also been used to analyze the signaling pathways of other glycosaminoglycans and their role in neuronal polarity. 62 Still in the cell migration area, the binding of HGF (hepatocyte growth factor) to its receptor (c-Met) leads to invasive growth, an essential developmental event also implicated in tumor metastasis. The first event in this cascade is receptor autophosphorylation. Peptides interfering with HGF-induced c-Met autophosphorylation coupled to penetratin, once internalized in normal and transformed epithelial cells, blocked c-Met kinase and invasive growth. 63 2.4.3.2 Delivery of Peptide Nucleic Acids (PNAs) PNAs are oligonucleotides in which the sugar-phosphate backbone has been replaced by a neutral peptidic backbone; 64 they bind complementary RNA and DNA in a parallel or antiparallel orientation. Compared to deoxy-oligonucleotides, they present the advantage of forming extremely stable PNA–DNA or PNA–RNA duplexes. Moreover, PNAs are highly resistant to proteases and nucleases and inhibit gene expression at transcriptional and translational levels. Unfortunately, PNAs are poorly internalized by live cells. Three reports have demonstrated that eucaryotic cells efficiently take up penetratin-coupled PNAs in vitro 65,66 and in vivo. 34,67 In vitro delivery of PNA using penetratin-1 was demonstrated in human prostate tumor cells 66 and human melanoma cell lines; 65 the aim was to investigate the effect
38 Cell-Penetrating Peptides: Processes and Applications of two inhibitory PNAs on the catalytic activity of human telomerase. Telomerase function in normal cells is to maintain chromosome integrity, but several reports raise the question of telomerase implication in cell immortality and cancer. From a therapeutic point of view, it is of interest to block telomerase activity in transformed cells. To add telomeric repeats to the end of chromosomes, telomerase copies a small RNA sequence complementary to DNA. The strategy used here was to internalize two PNAs, an 11mer and a 13mer designed to cover the 5′-proximal region of the RNA and thus to inhibit human telomerase activity. The 13mer PNA coupled to penetratin through a disulfide-bound down-regulated telomerase activity in intact live cells, but the doubling time of the treated cells was only slightly decreased and telomeres were not shortened. 65 A more successful use of PNAs linked to penetratin-1 was the antagonizing of the biological activity of galanin, a peptide neuromediator involved in pain transduction (see the section devoted to in vivo use of penetratin-derived peptides). 2.4.3.3 Delivery of Entire Proteins AntpHD and penetratin are able to deliver large molecular weight protein into live cells. Among them are homeoproteins like Engrailed, HoxA-5, HoxB-4, HoxC-8, Pax 6, HoxC-4, Emx2, and Otx2 68-70 (unpublished results). A good example of the successful delivery of entire proteins fused to penetratin-1 is the study of the replicative senescence phenomenon. Genetically programmed senescence is induced when cells approach their limit of population doubling. Interestingly, p16 INK4b RNA and protein levels rise as cells enter senescence, but there was no evidence that p16 accumulation drives senescence. Using penetratin-1 to internalize p16 INK4b into human diploid fibroblasts, Kato et al. have shown that p16 INK4b , but not a functionally compromised variant, provokes G1 arrest. 71 This arrest is certainly due to inhibition of pRb phosphorylation and the arrested cells display a senescent phenotype, strongly suggesting that p16 INK4b is implicated in replicative senescence. 2.4.3.4 Chemical Drug Delivery Doxorubicin, an anthracyclin, is a commonly used anticancer drug. Anthracyclins are among the most active antitumor compounds. Not only do they, as intercalating agents, induce DNA damage but they also bind cell surface acidic phospholipids, like cardiolipids, and induce necrosis and apoptosis via the sphingomyelin–ceramide pathway. However, anthracyclin treatments often lead to multidrug-resistant tumors through an enhanced expression of a multidrug-resistance gene (mdr). This gene encodes a 170-kD protein called the P-glycoprotein (Pgp) that actively transports several cytotoxic agents out of the cells. 72 To overcome this problem, Mazel et al. have covalently linked doxorubicin to the penetratin N terminus and tested this conjugate in human erythroleukemia K562 cells. 73 Doxo-penetratin was about 20-fold more effective than doxorubicin alone to kill K562 doxorubicin-resistant cells. This suggests that the mode of internalization of penetratin, very likely through inverted micelles, allows the conjugate to escape Pgp activity.
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Page 11 and 12: REFERENCES 1. Green, M. and Loewens
Page 14 and 15: Contributors Mats Andersson Microbi
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Page 18 and 19: Contents Section I Classes of Cell-
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Model Amphipathic Peptides 87 A cat
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Model Amphipathic Peptides 89 At th
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Model Amphipathic Peptides 91 16. H
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Signal Sequence-Based Cell-Penetrat
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116 Cell-Penetrating Peptides: Proc
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Interactions of Cell-Penetrating Pe
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Structure Prediction of CPPs and It
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FIGURE 9.7 (Color Figure 9.7 follow
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FIGURE 9.8 The center of the figure
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Biophysical Studies of Cell-Penetra
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Toxicity and Side Effects of Cell-P
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Kinetics of Uptake of Cell-Penetrat
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Cell-Penetrating Peptide Conjugatio
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Cell-Penetrating Peptides as Vector
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TABLE 16.1 Examples of Transport of
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CPP 2 Cargo or mRNA CAP Antisense A
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Microbial Membrane-Permeating Pepti
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Index A Abaecin, 129 Abz radiolabel
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Index 399 Diffraction, 168 Disulphi
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Index 401 structure prediction, 187
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Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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