Critical Region for Amyloid Fibril Formation of Mouse Prion Protein: Unusual Amyloidogenic Properties of the Helix 2 Peptide

Abstract

To gain insight into the structural mechanism of the conformational conversion process of prion, we examined the potential amyloidogenic property of each secondary structural element in a mouse prion protein (mPrP) and discriminated their relative significance for the formation of amyloid fibrils. Although peptides corresponding to alpha-helix 2 and alpha-helix 3 (named H2 peptide and H3 peptide, respectively) formed the amyloid-like fibrils, their structures were quite different. H2 fibrils formed the ordered beta-sheet with the beta-turn conformation, and the resultant fibrils were long and straight. In contrast, H3 fibrils consisted of the beta-sheet with the random conformation, and the resultant fibrils were short and flexible. These properties are basically consistent with their hydrophobicity and beta-strand propensity profiles. To examine the cross reactivity between peptide fragments and full-length mPrP, we then carried out seeding experiments. While H2 seeds induced the formation of fibrils of full-length mPrP as quickly as full-length mPrP seeds, H3 seeds exhibited a long lag time. This implies that the region of alpha-helix 2 rather than alpha-helix 3 in mPrP has great potential for initiating fibril formation. As a whole, the alpha-helix 2 region would be crucial for the nucleation-dependent replication process of the prion protein.

The role of the unusual threonine string in the conversion of prion protein

“…This local instability has been proposed as the underlying structural basis for initiating the PrP C -to-PrP Sc conversion24. Consistent with this hypothesis, the C-terminus of α-helix 2 has been found to be unstable2526, and the peptide corresponding to helix 2 is able to form amyloid fibrils that can act as seeds for full-length wild-type PrP27. Replacing threonines with α-helix-prone alanines stabilizes the PrP C structure and prevents in vitro PrP aggregation28.…”

Ultrasonication-Dependent Acceleration of Amyloid Fibril Formation

“…24,35,36 More recently, the effects of ultrasonication have been found to accelerate nucleation among several proteins and peptides. 19,23,[37][38][39][40] Interestingly, amyloid fibrils produced by ultrasonication were very short, with apparently similar lengths as determined by AFM (Fig. 1d), suggesting that short fibrils of homogeneous molecular sizes are formed efficiently with the opposing effects of fragmentation and nucleation.…”

Ultrasonication-dependent production and breakdown lead to minimum-sized amyloid fibrils

“…Although ultrasonication was originally used to prepare seeds from preformed fibrils (9), which were further applied to the amplification of infectious prion proteins (10,11), ultrasonication-dependent fragmentation is becoming an important approach to analyzing the properties of fibrils (12,13). However, its strong effect of agitation has recently been found to accelerate the fibril nucleation of several proteins and peptides (14)(15)(16)(17)(18). Interestingly, amyloid fibrils produced by ultrasonicationinduced fibrillation were very short with apparently similar lengths as determined by AFM (15,19), suggesting that short fibrils of homogeneous molecular size are formed efficiently under ultrasonication in combination with the opposing effects of fragmentation and of nucleation (Fig.…”