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84 Cell-Penetrating Peptides: Processes and Applications
indicate a promising basis for the development of vector peptides with increased
cell specificy.
Taken together, the CLSM results suggest that amphipathicity is without importance
for the translocation of peptides across mammalian plasma membranes, but
that it mediates enrichment within the cell interior and increases resistance against
wash-out. With a view to practical applications, therefore, substantial obstacles
connected with the use of amphipathic peptides, such as membrane toxicity, high
aggregation propensity, and related solubility problems, appear avoidable.
4.4 ABILITY OF MAPs TO SHUTTLE POLAR BIOACTIVE
COMPOUNDS INTO CYTOSOL AND NUCLEUS
OF MAMMALIAN CELLS
First attempts to assess the suitability of MAPs for introducing polar compounds of
biological interest into the cell interior of mammalian cells were performed using
disufide bridged conjugates of I with the negatively charged SH2 domain-binding
peptide pYEEWE and with the positively charged SV40 nuclear localization
sequence PKKKRKV. CLSM revealed internalization into the cytosol similarly to I
for both conjugates and enrichment within the nucleus for the conjugate bearing the
SV40 sequence. 10
Further experiments using the tripeptide SLV, the consensus sequence for the FAP-
1 binding domain of the intracellular C terminus of the apoptosis-inducing receptor
Fas, 34 and the polymerase II-inhibiting cyclic mushroom peptide amanitin 35,36 were
performed to examine the ability of such conjugates to elicit biological effects within
the cell after being tagged to I.
Human Fas (APO-1/CD95) is an apoptosis-inducing member of the tumor necrosis
factor receptor superfamily. 34,37 In the inactive state, a negative regulatory domain of
Fas is captured by Fas-associated-phosphatase-1 (FAP-1), 38 which blocks binding of
adaptors (e.g., FADD) and subsequent caspase activation. 34,39 Yanagisawa et al. recently
showed that cytoplasmic microinjection of the simple N-terminally protected tripeptide
Ac-SLV, the consensus sequence for the FAP-1 binding domain of Fas, inhibited the
Fas-FAP-1-binding and dramatically enhanced Fas-mediated apoptosis. 40 Figure 4.10
shows that externally administered I, bearing the tripeptide SLV C terminally, is able
specifically to activate caspase 8, the initial reaction of the apoptosis cascade. 41,42 The
specificity is indicated by the inactivity of the components alone and of the N terminally
with the SLV-sequence-tagged I (Figure 4.10; Reference 43). Replacement of I by the
natural Antennapedia sequence 9 resulted in similar biological activity (Figure 4.10),
indicating comparable shuttling ability for MAPs and natural vector peptide.
Disulfide bridging of I or the Antennapedia peptide 9 to the mushroom toxin
amanitin similarly augmented the toxicity of this cyclic peptide (Figure 4.11;
Reference 44), revealing the extensive and comparable shuttling ability of both cellpenetrating
peptides. For amanitin alone a tenfold higher concentration was required
for eliciting a toxicity comparable to that of its conjugates. 44