Transition of Amyloid Oligomers to Mature Fibrils: Internal Conversion Vs. Competing Assembly Pathways?

Abstract

Deposition of protein plaques, rich in long rigid fibrils with a characteristic cross-beta sheet structure, is the pathological marker for human disorders ranging from Alzheimer's disease to type II diabetes and rheumatoid arthritis. Significant evidence has implicated the formation of globular oligomeric amyloids as the main pathogenic agent in amyloid diseases. At the same time, in vitro experiments indicate that amyloid oligomers and rigid fibrils are formed along distinct assembly pathways with characteristic growth kinetics. This raises the questions how these early-stage oligomeric intermediates are converted to the rigid fibrils dominating during the late-stages of most amyloid diseases? We have investigated the transition from the formation of amyloid oligomers and their curvilinear polymers to the rigid late-stage fibrils using the model amyloid hen egg white lysozyme (hewL). We have shown that hewL oligomers form a distinct aggregate phase with a well-defined transition boundary. However these oligomers and their curvilinear fibrils are metastable against the formation of thermodynamically stable rigid fibrils. We therefore performed experiments to discern whether amyloid oligomer species were directly converted into the stable rigid fibril conformation or whether rigid fibril nucleation proceeded in parallel, i.e. in competition with, oligomer formation. To do so, we monitored the rates of rigid fibril nucleation right outside and inside the transition boundary for oligomer formation. Our data suggest that oligomer formation is in kinetic competition with rigid fibril nucleation for their monomeric growth substrate. Futhermore, we observed no signs that prior formation of oligomeric species accelerated the nucleation of rigid fibrils. The latter would be expected for conformational conversion of oligomers into rigid fibrils. If anything, prior formation of oligomers retards the nucleation of rigid fibrils.