Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation
Figure 5
CADA binds to the signal peptide of human CD4 and interacts primarily with its hydrophobic H-region.
(A) Binding of CADA to the SP of human but not to mouse CD4. Chemically synthesized peptides composed of the signal sequence and eight N-terminal residues of mature CD4, plus a PEG11 linker and a biotinylated lysine at the C-terminus, were captured on a streptavidin C1 chip for SPR analysis. The chip density was 84 and 153 resonance units (RUs) for human and mouse SP, respectively. Graph shows interaction of CADA (160 µM) to hCD4 SP (green) but not to mCD4 (blue). (B) As in (A), but for the binding of SRP to the SPs. The chip density was between 120 and 130 resonance units (RUs). Graph shows dose response of SRP (nM) to hCD4 SP (green). Similar binding of SRP (25 nM) was observed for mouse CD4 SP (blue). (C) The inactive CADA-analog MFS105 (MFS, red line) did not bind to the hCD4 SP (up to 1,000 µM), whereas for CADA a dose-dependent binding was measured (black lines). The chip density was 95 RUs. For clarity of the figure, only the 500 µM line is shown. Comparable data were obtained in an independent experiment shown in Figure S4D. (D) Schematic representation of the constructs used. The N-, H-, and C-regions of the SP of hCD4 were exchanged for those of mouse CD4. Also the first seven N-terminal residues of the mature protein were swapped as indicated. The residues of hCD4 are depicted in green, whereas those of mouse CD4 are in blue and underlined. (E) Flow cytometry analysis of HEK293T cells transiently transfected with the expression plasmids from (D) and left either untreated or treated with CADA for 48 h. Cells were collected and stained for hCD4. CD4 expression levels were normalized to non-treated controls (n≥3). The IC50 values of CADA for each construct are included.