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Signal Sequence-Based Cell-Penetrating Peptides 95 aimed at targeting the nucleus. 11,12 The nuclear transport process is facilitated by nuclear import signal sequences, the best documented group of which comprises the NLSs. NLSs are characteristic short sequences that can be further subdivided into monopartite or bipartite signals when they consist, respectively, of one or two clusters of four or more basic amino acids (lysine or arginine). 12-14 Understanding the nuclear transport mechanism of macromolecules, together with characterization of a large number of potential NLSs and their regulation, has enabled the development of new tools that can be directly applied to the improvement of drug delivery systems. In eukaryotic cells, most of the natural NLSs are bipartite, consisting of two short cationic domains separated by a spacer of 10 to 12 residues. The best characterized bipartite NLS is that of nucleoplasmin 15 with the minimal sequence KRPAATKKAGQAKKKL. Among monopartite NLS, the best characterized is a short sequence, 126 PKKKRKV 132 , derived from SV40 (simian virus 40) large T antigen. 16 Lys 128 is essential for NLS activity, as mutation of this residue to an Asn or a Thr completely abolishes nuclear targeting. 17 In contrast, mutation of the other basic residues is less dramatic for nuclear translocation. 11,18 This NLS has been extensively used for coupling cargoes (protein, peptide, DNA, or ODN) and has been shown to improve crossing of the nuclear membrane. 9,10 In addition to its cationic domains, NLS contain a few characteristic components, including an α-helix destabilizing proline or glycine residue upstream or downstream of the cluster of basic residues and a cluster of acidic residues upstream or downstream of or within the spacer. Serine residues located in or around the cationic cluster are usually essential for transport and have been implicated as phosphorylation sites. 9,20 Finally, hydrophobic aromatic residues (Trp or Tyr) are excluded from the NLS or from the spacer of the bipartite NLS. 5.2.2 MECHANISM OF NUCLEAR TRANSPORT The most widely utilized pathway for nuclear import is mediated by NLSs (Table 5.1 and Figure 5.1). Although there appears to be a lack of primary sequence homology between the different NLS sequences described, they all bind the same family of cytoplasmic receptors termed importins or karyopherins. The importin β pathway is one of the best described, with import substrates binding importin β either directly or through the adaptor protein importin α. 13,18-22 Transport of macromolecules between the cytoplasm and the nucleus generally occurs through the nuclear pore complex (NPC). The NPC is a large multimeric complex of about 125 MDa containing 50 to 100 different proteins, termed nucleoporins, 18,22 that forms an inner pore smaller than 9 nm in diameter. In theory molecules smaller than 9 nm in diameter or around 60 kDa can diffuse passively through the central channel of the NPC. However, small molecule delivery is also controlled by cellular events and other signals such as cytoplasmic retention signals and phosphorylation, which may block nuclear entry and render it more selective. In contrast, entry into the nucleus of larger molecules including proteins or DNA requires an active signal-mediated transport process facilitated by NLS signals. This nuclear transfer through the NPC involves a network of proteins and is dependent
96 Cell-Penetrating Peptides: Processes and Applications TABLE 5.1 NLS-Containing Cell-Penetrating Peptides Proteins Minimal NLS Ref. SV 40 Large T PKKKRKV 16 P101 CGPGSDDEAAADAQHAAPPKKKRKVGY 16, 17 Nucleoplasmin KRPAATKKAGQAKKKKL 15 HIV-1 Tat GRKKRRQRRRPPQ 42 Yeast mat a2 NKIPIKD 5 HIV-1 rev RRNRRRRW 42 Polyomavirus Vp1 APKRKSGVSK 38 NF-kB VQRKRQKLMP 10, 68 M9 NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY 29–31 Vpr DTWTGVEALIRILQQLLFIHFRIGCRHSRIGIIQQRRTRNGA 37, 38, 41 FP-NLS (MPG) GALFLGFLGAAGSTMGAWSQPKKKRKV 50, 51 SP-NLS MGLGLHLLVLAAALQGAWSQPKKKRK 47, 48 SN50 AAVALLPAVLLALLAPVQRKRQKLMP 10, 68 Ran-GTP GAP Ran-GDP α β NLS NLS α β Cytoplasm Nucleus Nuclear envelope FIGURE 5.1 Mechanism of NLS-dependent nuclear import pathway. The formation of the trimeric complex among an NLS containing cargo, importin α, and importin β takes place in the cytoplasm. This is followed by docking of the trimeric complex to the NPC, then translocation through the central channel, and finally Ran-GTP-mediated dissociation of the complex and release of the cargo into the nucleus. NLS α Ran-GTP NLS αα β β Ran-GTP GEF
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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The Tat-Derived Cell-Penetrating Pe
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24 Cell-Penetrating Peptides: Proce
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146 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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FIGURE 15.9 (Color Figure 15.9 foll
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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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