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Hydrophobic Membrane Translocating Sequence Peptides 117
The initial hypothesis proposed to answer questions such as whether there is a role
for the N-terminal extension synthesized on secretory proteins and whether it is
necessary for targeting was extended into a theory suggesting that signals exist to
initiate and to terminate translocation. 17 This seminal work resulted in the establishment
of the signal hypothesis and the 1999 Nobel Prize for Physiology or Medicine
for Blobel. Currently understood mechanisms used by proteins to translocate across
membranes are reviewed in Simon and Blobel. 18
6.3 HYDROPHOBIC TRANSLOCATING PEPTIDES BASED
ON THE SIGNAL SEQUENCE h-REGION
We now know that the signal sequence region consists of three distinct domains: a
hydrophilic N terminus, a hydrophobic core, and a polar C terminus that usually
defines a signal peptidase cleavage site. 19-22 The hydrophobic region (h-region) has
been shown to play a central role in the ability of the protein to penetrate the
membrane and, also, in the secondary conformation of the signal sequence. 23-28
Therefore, only the h-regions of certain signal sequences have been investigated as
translocation peptides; the flanking residues that do not tend to influence translocation
activity have been omitted. h-regions that generally range in length from 18 to
21 residues have been identified in over 100 proteins 29 and exhibit minimal primary
structure homology. It appears that the average hydrophobic character of the
h-region, and of the entire signal sequence, correlates more closely with observed
translocation activity than the actual amino acid sequence. 30-32 Considering the
translocating activity of the h-region peptides in the protein secretion process (protein
export), these peptides have been recently tested for their ability to translocate and
deliver cargo into living cells. 33 Lin et al. found that these h-region peptides are fully
capable of importing other functional peptides and proteins into living cells. 33-36
Therefore, these h-region-based peptides have been termed membrane translocating
sequence (MTS) peptides.
The following sections of this chapter describe the use of MTS peptides as
translocation peptides in living cellular systems. The applications of MTS peptides
in biological research and their potential in therapeutic development are also
reviewed.
6.3.1 DEVELOPMENT OF SN50 PEPTIDE: KFGF MTS LINKED TO NF-κB NLS
The hydrophobic MTS peptides are utilized most frequently for examination of
intracellular signal transduction pathways and protein trafficking. The h-region of
the signal sequence of Kaposi fibroblast growth factor (kFGF or FGF-4) was the
first such peptide tested as an MTS. 33 In one of the earliest experiments designed
to test the efficacy of the hydrophobic peptide, the 16-residue sequence AAVALL-
PAVLLALLAP from the h-region of kFGF was synthesized in tandem with a 25-residue
peptide representing a nonhydrophobic region of the kFGF protein. The complete
41-residue peptide with the h-region located at the N terminus was radiolabeled
through a tyrosine residue with 125 I and initially tested for uptake into cells in vitro.
The peptide, termed 125 I-SKP, was found to be associated with NIH 3T3 cells