High-throughput analysis of ultrasonication-forced amyloid fibrillation reveals the mechanism underlying the large fluctuation in the lag time

This research was originally published in the Journal of Biological Chemistry. Ayaka Umemoto, Hisashi Yagi, Masatomo So and Yuji Goto. High-throughput Analysis of Ultrasonication-forced Amyloid Fibrillation Reveals the Mechanism Underlying the Large Fluctuation in the Lag Time. J. Biol. Chem. 2014; 289, 27290–27299. © the American Society for Biochemistry and Molecular Biolog

Supersaturation-limited and unlimited phase transitions compete to produce the pathway complexity in amyloid fibrillation

Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.This research was originally published in the Journal of Biological Chemistry. Masayuki Adachi, Masatomo So, Kazumasa Sakura, József Kardos and Yuji Goto. Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation. J. Biol. Chem. 2015; 290, 18134-18145. © the American Society for Biochemistry and Molecular Biolog

Drastic acceleration of fibrillation of insulin by transient cavitation bubble

Nakajima K., Nishioka D., Hirao M., et al. Drastic acceleration of fibrillation of insulin by transient cavitation bubble. Ultrasonics Sonochemistry, 36, 206, 2016. https://doi.org/10.1016/j.ultsonch.2016.11.034

Time-Resolved Observation of Evolution of Amyloid-β Oligomer with Temporary Salt Crystals

The aggregation behavior of amyloid-β (Aβ) peptides remains unclarified despite the fact that it is closely related to the pathogenic mechanism of Alzheimer's disease. Aβ peptides form diverse oligomers with various diameters before nucleation, making clarification of the mechanism involved a complex problem with conventional macroscopic analysis methods. Time-resolved single-molecule level analysis in bulk solution is thus required to fully understand their early stage aggregation behavior. Here, we perform time-resolved observation of the aggregation dynamics of Aβ oligomers in bulk solution using liquid-state transmission electron microscopy. Our observations reveal previously unknown behaviors. The most important discovery is that a salt crystal can precipitate even with a concentration much lower than its solubility, and it then dissolves in a short time, during which the aggregation reaction of Aβ peptides is significantly accelerated. These findings will provide new insights in the evolution of the Aβ oligomer.Nakajima K., Yamazaki T., Kimura Y., et al. "Time-Resolved Observation of Evolution of Amyloid-β Oligomer with Temporary Salt Crystals", Journal of Physical Chemistry Letters, 11(15), 6176-6184, July 20, 2020. Copyright © 2020 American Chemical Society. https://doi.org/10.1021/acs.jpclett.0c01487

Mechanisms of ultrasonically induced fibrillation of amyloid β₁₋₄₀ peptides

We systematically study the relationship between the ultrasonically induced aggregation behavior of amyloid β₁₋₄₀ peptide and acoustic pressures to clarify the dominant mechanism of the aggregation. With ultrasonic irradiation, the thioflavin-T (ThT) level of the A solution rises after a lag time, takes a maximum at 5 h, and remains unchanged or decreases. Thus, we monitor the ThT level at 5 h to evaluate the progress of the β-sheet structure and investigate its correlation with the acoustic pressures of fundamental and harmonics waves. The second-harmonics-wave amplitude shows the highest correlation with the ThT level, indicating the dominant contribution of cavitation bubbles to the fibrillation phenomenon. The influence of solution pH and Ar gas are investigated to identify the aggregation mechanism. As a result, local condensation of the peptide due to the high affinity of hydrophobic residues to the bubble-solution interface causes a highly supersaturated solution, leading to precipitation of β-sheet-rich nuclei.Kentaro Uesugi, Hirotsugu Ogi, Masahiko Fukushima, Masatomo So, Hisashi Yagi, Yuji Goto and Masahiko Hirao. Mechanisms of ultrasonically induced fibrillation of amyloid β₁₋₄₀ peptides. Japanese Journal of Applied Physics, 52(7S), 07HE10. https://doi.org/10.7567/JJAP.52.07HE10

Optimized sonoreactor for accelerative amyloid-fibril assays through enhancement of primary nucleation and fragmentation

Nakajima K., Noi K., Yamaguchi K., et al. Optimized sonoreactor for accelerative amyloid-fibril assays through enhancement of primary nucleation and fragmentation. Ultrasonics Sonochemistry, 73, 105508, 2021. https://doi.org/10.1016/j.ultsonch.2021.105508

Optimized Ultrasonic Irradiation Finds Out Ultrastable Aβ₁₋₄₀ Oligomers

Oligomer species of amyloid β (Aβ) peptides are intensively investigated because of their relevance to Alzheimer's disease (AD), and a stable oligomer will be a cause of AD. In this article, we investigate the structural stability of two representative Aβ₁₋₄₀ oligomers, which are with and without the β-sheet structure, denoted by β and non-β oligomers, respectively, using optimized ultrasonic irradiation (OUI). Recent studies reveal that OUI significantly accelerates the fibril formation in Aβ₁₋₄₀ monomers; it is capable of transforming any unstable oligomers into fibrils (the dead-end products) in a short time. First, we find that β oligomers can be produced under high-speed stirring agitation; their β-sheet structures are evaluated by the circular-dichroism spectrum measurement, by the immunoassay using the fibril-specific OC antibody, and by the seeding experiment, showing identical characteristics to those formed in previous reports. Second, we form non-β oligomers in a high-concentration NaCl solution and confirm that they include no β-sheet structure, and they are recognized by the oligomer-specific A11 antibody. Furthermore, we confirm the neurotoxicity of the two types of oligomers using the neural tissue derived from mouse embryonic stem cells. We apply the OUI agitation to the β and non-β oligomers. The non-β oligomers are transformed into the fibrils, indicating that they are intermediate species in the fibrillation pathway. However, the β oligomers are surprisingly unaffected by OUI, indicating their high thermodynamic stability. We conclude that the β oligomers should be the independent dead-end products of another pathway, different from the fibrillation pathway.Kichitaro Nakajima, Masatomo So, Kazuma Takahashi, Yoh-ichi Tagawa, Masahiko Hirao, Yuji Goto, and Hirotsugu Ogi. Optimized Ultrasonic Irradiation Finds Out Ultrastable Aβ₁₋₄₀ Oligomers. Journal of Physical Chemistry B, 2017, 121(12), 2603-2613. ©2017 American Chemical Society. https://doi.org/10.1021/acs.jpcb.7b01409

Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation

This research was originally published in the Journal of Biological Chemistry. Ayame Nitani, Hiroya Muta, Masayuki Adachi, Masatomo So, Kenji Sasahara, Kazumasa Sakurai, Eri Chatani, Kazumitsu Naoe, Hirotsugu Ogi, Damien Hall, and Yuji Goto. Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation. J. Biol. Chem. 2017; 292, 21219-21230 © the American Society for Biochemistry and Molecular Biolog