Cytotoxicity of prion protein-derived cell-penetrating peptides is modulated by pH but independent of amyloid formation.

Prion diseases are associated with conversion of cellular prion protein (PrP) into an abnormally folded and infectious scrapie isoform (PrP). We previously showed that peptides derived from the unprocessed N-termini of mouse and bovine prion proteins, mPrP and bPrP function as cell-penetrating peptides (CPPs), and destabilize model membrane systems, which could explain the infectivity and toxicity of prion diseases. However, subsequent studies revealed that treatment with mPrP or bPrP significantly reduce PrP levels in prion-infected cells. To explain these seemingly contradictory results, we correlated the aggregation, membrane perturbation and cytotoxicity of the peptides with their cellular uptake and intracellular localization. Although the peptides have a similar primary sequence, mPrP is amyloidogenic, whereas bPrP forms smaller oligomeric or non-fibrillar aggregates. Surprisingly, bPrP induces much higher cytotoxicity than mPrP, indicating that amyloid formation and toxicity are independent. The toxicity is correlated with prolonged residence at the plasma membrane and membrane perturbation. Both ordered aggregation and toxicity of the peptides are inhibited by low pH. Under non-toxic conditions, the peptides are internalized by lipid-raft dependent macropinocytosis and localize to acidic lysosomal compartments. Our results shed light on the antiprion mechanism of the prion protein-derived CPPs and identify a potential site for PrP formation.