Stepwise Organization of the β- Structure Identifies Key Regions Essential for the Propagation and Cytotoxicity of Insulin Amyloid Fibrils

Background: Oligomers and protofibrils have been observed in the early stages of fibrillation. Results: Fibrillation of insulin at a high salt concentration identified a new species of prefibrillar intermediate. Conclusion: Structural comparison of the intermediate and mature fibrils suggested regions responsible for self-propagation and cytotoxicity. Significance: The trapping of intermediate is an effective way of revealing molecular details of the organization of fibril structure. Amyloid fibrils are supramolecular assemblies, the deposition of which is associated with many serious diseases including Alzheimer, prion, and Huntington diseases. Several smaller aggregates such as oligomers and protofibrils have been proposed to play a role in early stages of the fibrillation process; however, little is known about how these species contribute to the formation of mature amyloid fibrils with a rigid cross- structure. Here, we identified a new pathway for the formation of insulin amyloid fibrils at a high concentration of salt in which mature fibrils were formed in a stepwise manner via a prefibrillar intermediate: minute prefibrillar species initially accumulated, followed by the subsequent formation of thicker amyloid fibrils. Fourier transform infrared spectra suggested the sequential formation of two types of -sheets with different strength hydrogen bonds, one of which was developed concomitantly with the mutual assembly of the prefibrillar intermediate to form mature fibrils. Interestingly, fibril propagation and cellular toxicity appeared only after the later step of structural organization, and a comparison of -sheet regions between the prefibrillar intermediate and mature fibrils using proteolysis led to the proposal of specific regions essential for manifestation of these properties.