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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