Abstract
Non-fibrillar protein aggregates that appear in the earlier stages of amyloid fibril formation are sometimes considered to play a key role in amyloid nucleation; however, the structural features of these aggregates currently remain unclear. We herein identified a characteristic pathway of fibril formation by human insulin B chain, in which two major species of prefibrillar aggregates were identified. Based on the time-resolved tracking of this pathway with far-UV circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and 1H-NMR spectroscopy, the first prefibrillar aggregate with a hydrodynamic diameter of approximately 70 nm accumulated concomitantly with the formation of a β-sheet structure, and the size further evolved to 130 nm with an additional structural development. These prefibrillar aggregates were metastable and survived at least 24 hours as long as they were maintained under quiescent conditions. The energy barrier for nucleation was overcome by shaking or even by applying a single short ultrasonic pulse. Furthermore, an investigation where nucleation efficiency was monitored by fibrillation rates with varying the timing of the ultrasonic-pulse treatment revealed that the second prefibrillar aggregate specifically produced amyloid nuclei. These results suggest that the second form of the prefibrillar aggregates acts as a direct precursor for the amyloid nucleation.
Highlights
Based on the linear relationship between the amount of the 2nd prefibrillar intermediate and that of the nuclei produced upon the ultrasonic treatment (Fig. 6d), the nucleation of insulin B chains occurs via a multistep scheme, not a simple one-step one
No trace of conformational changes during nucleation was detected in the present study, the instant formation of amyloid nuclei after the ultrasonic pulse suggests that nucleation proceeded within the 2nd prefibrillar intermediate following a nucleated-conformational-conversion (NCC) mechanism, in which early aggregates are directly converted to amyloid nuclei
Previous finding showed that aggregates convert to mature amyloid fibrils in parallel with their mutual association[35], which may be categorized as the NCC mechanism in a broad context
Summary
Based on the linear relationship between the amount of the 2nd prefibrillar intermediate and that of the nuclei produced upon the ultrasonic treatment (Fig. 6d), the nucleation of insulin B chains occurs via a multistep scheme, not a simple one-step one. No trace of conformational changes during nucleation was detected in the present study, the instant formation of amyloid nuclei after the ultrasonic pulse suggests that nucleation proceeded within the 2nd prefibrillar intermediate following a nucleated-conformational-conversion (NCC) mechanism, in which early aggregates are directly converted to amyloid nuclei. Since its first proposal in 200031, the NCC mechanism has been supported by studies demonstrating the presence of on-pathway prefibrillar intermediates[5,8,9,32]. Previous finding showed that aggregates convert to mature amyloid fibrils in parallel with their mutual association[35], which may be categorized as the NCC mechanism in a broad contex
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
