Establishment of a prion protein (PrP) aggregation assay and characterization of PrP aggregates

Abstract

Mechanism underlying prion propagation is yet an enigmatic biochemical event. Prion propagation is based on the conformational conversion of the host cellular prion protein (PrPC) to the pathogenic isoform, termed PrPSc. During this event, α-helix-rich monomer of PrPC is converted into an aggregation-prone isoform with high β-sheet content. PrPSc becomes aggregated and spontaneously forms fibril structures in certain cases. Despite the fact that PrP aggregation is highly associated with the progression of prion diseases, understandings of their occurrence and function are limited and much remains controversial. Studies of growth and propagation of these aggregates require an optimized in vitro assay. To understand details on formation of PrP aggregates, various conditions that influence the process in vitro was investigated. This thesis describes such PrP aggregate formation to be influenced by denaturing conditions, thioflavin T (ThT) concentration, and PrP concentration. Recombinant PrPs of different species with different truncations affected effectual generation of aggregates, while the aggregating pattern over time was conserved. Generated aggregates were insoluble in detergent-free buffer and remained stable under non-freezing temperatures. Aggregates were sensitive to proteinase K, and high molecular weight 2 aggregates were not observed in semi-denaturing gel electrophoresis. Transmission electron micrographs demonstrated elongated as well as amorphous PrP aggregate structures. In this study, an assay is established to recapitulate aggregation of PrP and the outcome of the assay was characterized. These results support the idea that PrP aggregates analyzed in this study may represent those in the early stages of fibril formation.