Replacement Of Residue H95 With Charged Amino Acids In The Prion Protein Decreases Prion Conversion Propensity

Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders caused by a change in conformation of the prion protein from the normal cellular form (PrPC) to a misfolded form (PrPSc). Prion diseases have been widely studied, but despite many great leaps in our knowledge many gaps in our knowledge are still unknown, especially in prion conformational conversion mechanism. In this work, we perform an amino acid scan at histidine (H)95, a key regulator residue within proposed prion conversion of the fifth copper-binding site. We created a series of mutant PrP by replacing H95 with every other common amino acid and compared the prion conversion propensity by ScN2a assay. The results are remarkable with the residues with electrically-charged side chains (H95D, H95E, H95K and H95R) decreasing prion conversion by about 50-75%, compared to wild-type (wt) PrP. We next analyze the localization, trafficking and biochemical features of PrP H95E, the most promising mutant for reducing conversion, in N2aPrP(-/-) cells stably transfected with the mutant PrP. The only difference observed was relative to the endosomal recycling compartments, with mutant PrP H95E showing less co-localization compared to WT PrP. This is in agreement with other work that links the endosomal recycling compartments with prion conversion. We conclude that the replacement of PrP H95 with electrically charged side chains decreases the prion conversion propensity, and this is likely due to reduced co-localization with the endosomal recycling compartments.