Peptide Aptamers Expressed in the Secretory Pathway Interfere with Cellular PrP Sc Formation

Abstract

Prion diseases are rare and obligatory fatal neurodegenerative disorders caused by the accumulation of a misfolded isoform (PrP Sc) of the host-encoded prion protein (PrP c). Prophylactic and therapeutic regimens against prion diseases are very limited. To extend such strategies we selected peptide aptamers binding to PrP from a combinatorial peptide library presented on the Escherichia coli thioredoxin A (trxA) protein as a scaffold. In a yeast two-hybrid screen employing full-length murine PrP (aa 23–231) as a bait we identified three peptide aptamers that reproducibly bind to PrP. Treatment of prion-infected cells with recombinantly expressed aptamers added to the culture medium abolished PrP Sc conversion with an IC 50between 350 and 700 nM. For expression in eukaryotic cells, peptide aptamers were fused to an N-terminal signal peptide for entry of the secretory pathway. The C terminus was modified by a glycosyl-phosphatidyl-inositol-(GPI) anchoring signal, a KDEL retention motif and the transmembrane and cytosolic domain of LAMP-I, respectively. These peptide aptamers retained their binding properties to PrP c and, depending on peptide sequence and C-terminal modification, interfered with endogenous PrP Scconversion upon expression in prion-infected cells. Notably, infection of cell cultures could be prevented by expression of KDEL peptide aptamers. For the first time, we show that trxA-based peptide aptamers can be targeted to the secretory pathway, thereby not losing the affinity for their target protein. Beside their inhibitory effect on prion conversion, these molecules could be used as fundament for rational drug design.