crc press - E-Lib FK UWKS

98 Cell-Penetrating Peptides: Processes and Applications
combined with other transfection methods to facilitate delivery into the nucleus (see
Table 5.1).
Most of these studies were performed with the sequence derived from SV40
large T antigen NLS PKKKRKV. This sequence was associated with either membrane-penetrating
peptides or cationic peptides, but also directly linked to cargoes. 5,8,9
Several studies were performed with an optimized NLS sequence derived from SV40
large T antigen NLS (peptide P101) CGPGDDEAAADAQHAAPPKKKRKVGY,
which contains the NLS signature PKKKRKV together with the flanking serine
phosphorylation site S 112 DDE recognized by the protein kinase CK2. 32 This phosphorylation
was shown to improve binding of the NLS sequence to a receptor such
as importin α and consequently to enhance the rate of nuclear import. In peptide
P101, a combination of this NLS with polylysine enabled the demonstration of a
direct correlation among the presence of the NLS, recognition by the nuclear import
machinery, and the enhancement of transfection efficiency. 17,33,34
The NLS sequence M9 is a cellular NLS from heterogeneous nuclear ribonucleoprotein
hnRNP A1, a major nuclear pre-mRNA-binding protein. The sequence of M9
constitutes a nonclassical NLS, NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAK-
PRNQGGY, which is recognized by the importin β like shuttle protein transportin I. 35
Nuclear import based on the use of M9 sequence is extremely efficient and has been
resorted to in order to improve nuclear delivery of drugs and DNA. 30,31,36
Viruses achieve nuclear entry mostly during mitosis. However, several viruses
succeed in entering the nucleus through interaction of viral components with cellular
importin molecules. Although this mechanism is not well described, a few nonclassical
NLSs have been proposed to be involved and have further been used for nuclear
delivery. 37 Adenoviruses require dissociation of their viral components prior to entry
into the nucleus. In contrast, in the case of lentiviruses, the preintegration complex
enters the nucleus intact. 38 The protein Vpr is involved in the nuclear import of the
HIV preintegration complex and contains a nonclassical NLS at its C terminus,
DTWTGVEALIRILQQLLFIHFRIGCRHSRIGIIQQRRTRNGA, that allows nuclear
import to be mediated independently of the importin or transportin pathways. Vpr
has been proposed to interact directly with the NPC in an energy-independent
fashion. The NLS derived from the HIV viral protein Vpr has been used successfully
in transfection experiments. 37 The C-terminal domain of Vpr (Vpr 52-96 ) has been
shown to interact with and condense DNA and to mediate DNA transfection through
the endosomal pathway, but in a pH-independent manner. 39-41
A large number of NLSs or putative NLSs have been described, such as the
putative NLS found in the amino terminus of yeast α-mating factor, Q-KIPIK-N,
which is capable of enhancing delivery of beta galactosidase into the nucleus. More
recently, the HIV-1 Tat peptide or HIV Rev has also been proposed to represent an
NLS domain recognized directly by importin β rather than by importin α. 42 Tat
peptide can play a double targeting role, by first delivering its cargo into the cytosol,
then to the nucleus. A peptide sequence issued from the 20 first residues of the
adenovirus fiber protein was used to improve transfection of human cells. This
peptide AKRARLSTSFNPVYPYEDES contains a hydrophobic domain FNPVYPY,
specific for receptor-mediated endocytosis and the NLS motif KRARLSTSF, which
together favor delivery of proteins into the nucleus. 38,43