title: Stepwise organization of the β-structure identifies key regions essential for the propagation and cytotoxicity of insulin amyloid fibrils.
abstract: 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-β 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 β-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 β-sheet regions between the prefibrillar intermediate and mature fibrils using proteolysis led to the proposal of specific regions essential for manifestation of these properties.
authors.affiliation: Department of Chemistry, Graduate School of Science, Kobe University, Hyogo 657-8501, Japan. Electronic address: chatani@crystal.kobe-u.ac.jp.
authors.forename: Eri
authors.initials: E
authors.lastname: Chatani
authors.affiliation: College of Pharmaceutical Sciences, Ritsumeikan University, Shiga 525-8577, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Imamura
authors.affiliation: College of Pharmaceutical Sciences, Ritsumeikan University, Shiga 525-8577, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: College of Pharmaceutical Sciences, Ritsumeikan University, Shiga 525-8577, Japan. Electronic address: kato-m@ph.ritsumei.ac.jp.
authors.forename: Minoru
authors.initials: M
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D006358:Hot Temperature; D006860:Hydrogen Bonding; D007328:Insulin / Q000737:chemistry*; D013058:Mass Spectrometry; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D016716:PC12 Cells; D017433:Protein Structure, Secondary; D051381:Rats; D012492:Salts / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013997:Time Factors
chemical_list: D000682:Amyloid; D007328:Insulin; D012492:Salts; D012965:Sodium Chloride
keywords: Amyloid; Cytotoxicity; Insulin; Prefibrillar Intermediate; Propagation; Protein Aggregation; Protein Misfolding; Protein Structure