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Penetratins 31 2.4 DELIVERY OF HYDROPHILIC CARGOES INTO LIVE CELLS WITH PENETRATIN VECTORS Since first reports demonstrating the efficient cell delivery of hydrophilic molecules linked to penetratin or AntpHD, 31-33 several applications have been developed thanks to the Antennapedia homeodomain, its third helix, and several variants (for reviews see References 28, 29, 34, and 35). Penetratin and penetratin-derived peptides have been used successfully to deliver chemical molecules, proteins, oligopeptides, oligonucleotides, and peptide-nucleic acids into live cells, in vitro and in vivo. The next paragraphs describe the mode of linkage and highlight some typical applications. A more comprehensive list of cargoes delivered by this vector system is presented in Table 2.3. 2.4.1 PRINCIPLES OF CARGO–VECTOR LINKAGE One of the first applications has been to link and internalize oligonucleotides bearing a free thiol group to the cysteine naturally present in the β-turn between the second and the third helix of AntpHD. 36 AntpHD was also used to deliver recombinant polypeptides of different lengths (Table 2.3). In that case, the cargo sequences were fused to the C terminus of AntpHD and the fusion proteins expressed in Escherichia coli were purified thanks to the heparin-binding properties of the homeodomain. However, most of the experiments were achieved with the penetratin-1 peptide. The three main types of coupling protocols used are illustrated in Figure 2.2. In a first protocol, penetratin is synthesized with an additional N-terminalactivated cysteine, protected by a nitro-pyridinium group that prevents peptide homodimerization and facilitates disulfide bond formation with the reactive thiol present on the cargo. This method is advantageous in that the cargo is released free in the cytoplasm as a result of disulfide bond breakage in the reductive cytoplasmic milieu. In a second method, the cargo and vector are chemically synthesized in continuity. In that case, no coupling reaction is necessary. These first two methods also allow the internalization of modified peptides like phospho-peptides or peptide nucleic acid (PNA). Another possible modification is biotinylation; this modification is useful to follow the peptide in the cells and to purify any molecule interacting with internalized cargoes. The third method consists in the preparation of fusion polypeptides by in vitro recombination. The fusion is expressed in E. coli and purified easily due to the heparin-binding properties of the third helix. Alternatively, the fusion protein can incorporate a poly-histidine or GST sequence for purification on ion columns or gluthathion, respectively. Indeed a tag (HA, myc, etc.) and a protease site allowing removal of the GST moiety can be added to the construction. Because of low cost, this is a method of choice when long cargoes are needed. 2.4.2 VECTORIZATION WITH ANTPHD 2.4.2.1 AntpHD-Mediated Internalization of Oligonucleotides The first applications were developed with the entire homeodomain. AntpHD has been used to internalize antisense oligonucleotides against the β-amyloid precursor
32 Cell-Penetrating Peptides: Processes and Applications TABLE 2.3 Cargoes Internalized In Vitro and In Vivo with AntpHD-Derived Peptidic Vectors Type of cargo Length Vector Cargo effect Ref. Entire protein p16 INK4A 156 aa Penetratin Induces replicative senescence 71 Oligopeptide PKC 14 aa AntpHD Inhibits PKC activity in neurons 31 PKC-ε 9 aa Penetratin Inhibits PKC-ε activity in rabbit cardiomyocytes 58 cGPK 8 aa Penetratin Inhibits cGMP kinase in cerebral arteries 59 SSeKS 16 aa Penetratin Inhibits SSeKS binding to cyclinD1 51 ICE 6 aa Penetratin Blocks ICE proteases 43 EGF-R 9 aa Penetratin Inhibits EGF and PDGF stimulated mitosis 39 FGF-R1 9 aa Penetratin Inhibits FGF-R signaling in neurons 60 p75 NTR 35 aa Penetratin Suppresses neuronal death promoted by the chopper domain of p75 NTR 82 Pcw3 41 aa AntpHD T-cell activation 40, 41 OVA-K 8 aa Penetratin T-cell activation and protection against ovalbumin expressing tumor cell line 74 Rab 13 41 aa AntpHD Inhibits prolactin exocytosis 33 Grb10 16 aa Penetratin Blocks PDGF and insulin-stimulated mitogenesis 38 PSM 16 aa Penetratin Blocks PDGF and insulin-stimulated mitogenesi 39 Cav1 19 aa Penetratin Inhibits NO synthesis and inflammation 75 Met-R 19 aa Penetratin Inhibits Met kinase activity 63 CD44 13 aa Penetratin Inhibits CD44 activation 61 p16 INK4A 20 aa Penetratin Inhibits cyclin D1-CDK4 activity 45, 46 p16 INK4A 20 aa Penetratin Inhibits cyclin D1-CDK4 mediated cell-spreading on vitronectin 48 p16 INK4A 20 aa Penetratin Inhibits transformation of pancreatic tumor cells 47 p21 Waf1/Cip1 20 aa Penetratin Inhibits PCNA activity and promotes growth arrest of p53 –/– cells 80 p21 Waf1/Cip1 26 aa Penetratin Inhibits cyclin D1/CDK4 and cyclin E/CDK2 activity and kills human lymphoma cells 83 elF4e 12 aa Penetratin Blocks elF4e-induced transformation 52 p53/E1B 17 aa Penetratin Inhibits E1B binding to p53 53 p53 95 aa Penetratin Inhibits p53-mediated apoptosis 54 cMyc 14 aa Penetratin Inhibits cancerization of human breast cancer cells 56, 57 E2F-1 8 aa Penetratin Inhibits cyclin A/E-CDK2 activity 49 Oligonucleotide SOD1 21 mer Penetratin Inhibits SOD1 in PC12 cells 42 β-APP 25 mer AntpHD Inhibits β-APP in neurons 32 p21 Waf1/Cip1 21 mer AntpHD Inhibits p21 Waf1/Cip1 in human gliomas 37 HAND 18 mer Penetratin Inhibits the bHLH factor HAND in neural crest cells 81
Page 2 and 3: CELL- PENETRATING PEPTIDES Processe
Page 4 and 5: Pharmacology and Toxicology: Basic
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Page 14 and 15: Contributors Mats Andersson Microbi
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Model Amphipathic Peptides 81 relat
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Structure Prediction of CPPs and It
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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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