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74 Cell-Penetrating Peptides: Processes and Applications
pmol/mg protein
160
140
120
100
80
60
40
20
0
37°C
0°C
I all-D-I XV
FIGURE 4.2 Intact fractions of cell-associated peptide after exposing LKB Ez 7 cells for
30 min at 37 and at 0°C to 1.8 µM I, the all-D enantiomer of I, and the Antennapedia peptide
XV 9 and subsequent treatment with diazotized 2-nitroaniline. Before exposure to the peptide
at 0°C the cells were incubated in DPBSG for 60 min at 0°C. Each bar represents the mean
of three samples ± SD.
essential for this type of peptide to enter mammalian cells; this was modified later,
in that amphipathicity mediates only retrieval within the cell, combined with reduced
efflux (see below). Likewise, the inability to detect cellular uptake of II also argues
against a significant contribution from endocytosis to the internalization of I. If
adsorptive endocytosis were to be the predominant mechanism for the uptake, then
peptide II should also be internalized to a clearly measurable degree, since this
derivative, which possesses the same number of positive charges as I, has been found
to be adsorbed extensively to the cell surface (to about 30% to that of cell-associated I). 8
4.2.3 EFFECTS OF FACTORS KNOWN TO AFFECT ENDOCYTOSIS OR TRANSPORT
PROTEIN FUNCTION ON THE CELLULAR UPTAKE OF I
Figure 4.3 shows the effects of energy depletion, lowered temperature, pH, and ionic
composition of the incubation buffer, and of additives known to affect endocytosis
or to inhibit mdrp or MRP and the large anion transporter, respectively, upon
internalization of I into aortic endothelial cells. With the exception of calcium
depletion and vincristine treatment, the total quantity of internalized I was affected
significantly in all cases. Potassium depletion (known to inhibit clathrin-dependent
endocytosis 15,16 ) elevation of lysosomal pH by 100 µM chloroquine, 17 disturbance
of endosomal transport by 5 µM Brefeldin A, 18 addition of DNP-S-glutathione
(100 µM) (an inhibitor of MRP and the large anion transporter MOAT 19,20 ), and
depletion of magnesium had no effect upon internalization of I. 10