Cytotoxicity of Prion Protein-Derived Cell Penetrating Peptides is Independent of Amyloid Formation

Abstract

Prion diseases are associated with the conversion of the benign cellular form of the prion protein (PrP(C)) into an abnormally folded and aggregated scrapie isoform (PrP(Sc)), which is also responsible for prion infectivity. We previously showed that peptides derived from the unprocessed N-termini of mouse and bovine prion proteins, mPrP(1-28) and bPrP(1-30), function as cell-penetrating peptides (CPPs) that can efficiently deliver a whole host of cargos. mPrP(1-28) and bPrP(1-30) also exhibited membrane-perturbation effects in model membrane systems. Taken together, the behavior of mPrP(1-28) and bPrP(1-30) provides a potential mechanism for the infectivity and toxicity associated with prion diseases. However, in subsequent studies treatment with mPrP(1-28) or bPrP(1-30) significantly reduced PrPSc levels in prion-infected cells and substantially prolonged the time before infection was manifested after infecting healthy cells with scrapie. To explain these seemingly contradictory results, we correlated the aggregation and toxicity of mPrP(1-28) and bPrP(1-30) with their cellular uptake and intracellular localization. Although the two peptides have a similar primary sequence, mPrP(1-28) was highly prone to aggregation and formed amyloid fibers, whereas bPrP(1-30) aggregated much less and was non-amyloidogenic. Surprisingly, the non-amyloidogenic bPrP(1-30) induced much higher cytotoxicity than the amyloidogenic mPrP(1-28), suggesting that amyloid formation and toxicity are independent. The toxicity of these peptides was due to membrane perturbation. Interestingly, aggregation and toxicity of the peptides were inhibited by low pH. Following internalization by lipid-raft dependent macropinocytosis, a receptor-independent form of endocytosis, the peptides localized to lysosomes, where aggregation and toxicity were inhibited. Our results shed light on the antiprion mechanism of the peptides and provide a potential site for PrP(Sc) formation.