Study of a signal peptide-specific translocation inhibitor: a unique down-modulator of CD4

Signal peptides are crucial in the biogenesis of eukaryotic proteins for the selective routing and delivery of secreted and membrane-bound proteins towards the endoplasmic reticulum. However, the specific interactions between these targeting signals and the other cellular components that facilitate protein translocation and membrane integration are not yet fully understood. Previously, our lab discovered a small molecule called CADA which specifically inhibits the synthesis and export of human CD4, an important membrane glycoprotein in immune cells and the main receptor for the HIV virus. Recently, our lab could link the down-modulating activity of CADA to the signal peptide of CD4 that led to selective inhibition of its co-translational translocation. This discovery creates a unique opportunity to investigate the molecular events that occur during the early stages of protein translocation. The goal of this project was to develop an in-depth model of the interaction between a chemical compound (CADA), signal peptides and the complex cellular protein translocation machinery. Signal peptides have diverse amino acid sequences and therefore offer an interesting target for selective regulation of protein translocation. Our model will expand the current understanding of signal peptide functioning and will allow us to develop an efficient screening model for the exploration of additional candidate molecules and signal peptide libraries. We expect these results to impact the development of innovative antiviral and medicinal applications. First, I contributed to a systematic analysis of CADA’s mode of action. The amino-terminal region of the CD4 pre-protein, which includes the signal peptide, is necessary and sufficient for CADA sensitivity. The compound selectively interacts with this signal peptide and thereby inhibits co-translational translocation of CD4 pre-proteins across the endoplasmic reticulum membrane. Affected pre-protein chains incorrectly enter the cytosol, and become degraded by the proteasome. In accordance with one of the current signal peptide topology models, CD4 initially enters the translocation channel in a ‘head-on’ orientation but its topology inverts at a certain length, to form a loop and to allow further translocation. CADA prevents this mandatory inversion and thereby blocks subsequent translocation of the chain. The inhibitory effect of CADA appeared to be selective for CD4, so I aimed to identify additional targets of the compound in the next step. Using a PowerBlot Western array, I evaluated the abundance of over 400 proteins in CADA-treated SUP-T1 cells and could identify sortilin as an additional substrate. CADA reduces sortilin expression by inhibition of the co-translational translocation in a signal peptide-dependent way, as is the case with CD4. I also described the CD4 down-modulation and antiviral activity of 28 new CADA analogs. This quantitative structure-activity relationship study identified a correlation between the arenesulfonyl side arm dipole moment and the biological compound activity. Additionally, analog CK147 showed the highest potency of all analogs recorded to date, and was over 6 times more active compared to CADA. Finally, I refined our model of CADA’s action, by determining the contribution of individual amino acids in the CD4 signal peptide towards CADA sensitivity with systematic mutagenesis. Two key residues in the CD4 signal peptide, Gln-15 and Pro-20, were identified as critical for CADA action. Additionally, positively charged residues in the mature protein domain that lie directly C-terminal to the signal peptide cleavage site are also required for full sensitivity. However, analysis of the sortilin signal peptide suggested that primary structure information alone is not sufficient to predict CADA sensitivity, as a specific three-dimensional positioning of key residues in the signal peptide and subsequent mature protein domain is likely required for CADA action.status: publishe