crc press - E-Lib FK UWKS

Hydrophobic Membrane Translocating Sequence Peptides 119
6.3.2 APPLICATIONS OF SN50 PEPTIDE
The peptide SN50 is now used as a general reagent in molecular biology due to its
proven ability to prevent NF-κB-induced gene expression. Kilgore et al. 41 used SN50
in a study of the role of NF-κB in the up-regulation of certain proteins during
formation of the membrane attack complex (MAC) of complement. Following activation
of the complement cascade, the assembly of the MAC induces production of
inflammatory mediators, e.g., IL-8, monocyte chemoattractant protein-1 (MCP-1);
however, the mechanism by which this occurred was unknown. Through a detailed
assessment of the nuclear activity of NF-κB in the presence and absence of SN50
following MAC formation, the authors concluded that MAC formation may serve
to activate IL-8 and MCP-1 genes through activation and translocation of NF-κB.
In a second example of SN50 used as a general reagent, the peptide was
employed to determine the role of NF-κB in the response of myocardial tissue to
ischemia. Maulik et al. 42 speculated that NF-κB was involved in myocardial adaptation
to ischemia. In this study rat hearts were rapidly excised and retrograde
perfusion established before subjecting the hearts to ischemic stress and monitoring
the adaptation. To define the role of NF-κB, the SN50 peptide was added to the
perfusion buffer and hearts were exposed to 18 µM of the peptide over a 15-min
period. It was found that the beneficial effects of ischemic adaptation were blocked
by pretreating the hearts with SN50. This shows that SN50 is capable of translocating
not only into cell suspensions but also into tissue.
More examples of the recent uses of SN50 to delineate the role of NF-κB in
signaling pathways are listed in Table 6.1. Results presented in these studies vary;
in some cases NF-κB was found to be important in the pathway examined, but in
other situations it has been determined that NF-κB is not involved.
6.3.3 KFGF MTS AIDS IN TRANSLOCATION OF OTHER CARGOES
Based on early success of the delivery of NF-κB NLS into intact cells when
covalently linked to the kFGF MTS, Lin et al. 34 proceeded to link the same MTS to
a different NLS, this time derived from the protein fibroblast growth factor-1
(FGF-1). The purpose of this assembly was to produce a reagent allowing examination
of the functional role of the NLS in FGF-1-stimulated DNA synthesis and
mitogenesis. A stepwise solid-phase peptide synthesis method was employed to
produce this chimeric peptide (SA), with the MTS located at the N terminus separated
from the NLS by a three-residue spacer (AAA). An indirect immunofluorescence
assay was used to show that SA peptide migrated into NIH 3T3 cells with
localization to the perinuclear areas and a thymidine incorporation assay showed
that the peptide induced a mitogenic response in a manner similar to the entire FGF-1
protein. A peptide representing the NLS alone was not capable of inducing such a
response and it was therefore concluded that the entire SA peptide was required for
cell penetration and biological activity. This same peptide was also found to be
effective in a range of other cell types, i.e., BALB/c3T3 murine fibroblasts, human
vascular endothelial cells, and primary cultured hepatocytes. 74