Masatomo So

Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation

Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two forms are often treated indiscriminately. β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins.

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

Background: Relationship between amyloid fibrils and amorphous aggregates has not yet been elucidated. Results: A competitive mechanism of amyloid fibrillation and amorphous aggregation reproduced the observed aggregation kinetics of β2-microglobulin. Conclusion: Apparent complexities in amyloid fibrillation are explained assuming supersaturation-limited crystal-like amyloid fibrils and unlimited glass-like amorphous aggregates. Significance: Linkage of the kinetics of protein aggregation and a conformational phase diagram improves the understanding of protein aggregation. 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.

Ultrasonication: An Efficient Agitation for Accelerating the Supersaturation-Limited Amyloid Fibrillation of Proteins

Amyloid fibrils are self-assemblies of proteins with an ordered cross-β architecture. Because they are associated with serious disorders, understanding their structure and mechanism of fibrillation is important. Irradiation with ultrasonication leads to fragmentation of amyloid fibrils, useful for seeding experiments. Recently, ultrasonication has been found to trigger the spontaneous formation of fibrils in solutions of monomeric amyloidogenic proteins. The results indicate that amyloid fibrillation is similar to the crystallization of solutes from a supersaturated solution. The accelerating effects of ultrasonication on amyloid fibrillation suggest that cavitation microbubbles play a key role in effectively converting the metastable state of supersaturation to the labile state, leading to spontaneous fibrillation. Moreover, ultrasonic irradiation would be promising for a high-throughput screening assay of amyloid fibrillation, advancing the study of supersaturation-limited amyloidogenesis.

A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.

Revisiting supersaturation as a factor determining amyloid fibrillation

Amyloid fibrils involved in various diseases are formed by a nucleation-growth mechanism, similar to the crystallization of solutes from solution. Solubility and supersaturation are two of the most important factors determining crystallization of solutes. Moreover, crystallization competes with glass formation in which solutes collapse into amorphous aggregates. Recent studies on the formation of amyloid fibrils and amorphous aggregates indicate that the partition between distinct types of aggregates can be rationally explained by a kinetic and thermodynamic competition between them. Understanding the role of supersaturation in determining aggregation-based phase transitions of denatured proteins provides an important complementary point of view to structural studies of protein aggregates.

High-throughput Analysis of Ultrasonication-forced Amyloid Fibrillation Reveals the Mechanism Underlying the Large Fluctuation in the Lag Time

Background: Ultrasonication effectively breaks supersaturation and forces amyloid fibrillation. Results: A high-throughput analysis of amyloid fibrillation showed that, although the lag time varied depending on the conditions, its coefficient of variation was constant. Conclusion: The large fluctuation in the lag time originates from a process associated with a common amyloidogenic intermediate. Significance: High-throughput analysis is powerful enough to clarify the mechanisms of supersaturation-limited phase transitions of proteins. Amyloid fibrils form in supersaturated solutions of precursor proteins by a nucleation and growth mechanism characterized by a lag time. Although the lag time provides a clue to understanding the complexity of nucleation events, its long period and low reproducibility have been obstacles for exact analysis. Ultrasonication is known to effectively break supersaturation and force fibrillation. By constructing a Handai amyloid burst inducer, which combines a water bath-type ultrasonicator and a microplate reader, we examined the ultrasonication-forced fibrillation of several proteins, with a focus on the fluctuation in the lag time. Amyloid fibrillation of hen egg white lysozyme was examined at pH 2.0 in the presence of 1.0–5.0 M guanidine hydrochloride (GdnHCl), in which the dominant species varied from the native to denatured conformations. Although fibrillation occurred at various concentrations of GdnHCl, the lag time varied largely, with a minimum being observed at ∼3.0 m, the concentration at which GdnHCl-dependent denaturation ended. The coefficient of variation of the lag time did not depend significantly on the GdnHCl concentration and was 2-fold larger than that of the ultrasonication-dependent oxidation of iodide, a simple model reaction. These results suggest that the large fluctuation observed in the lag time for amyloid fibrillation originated from a process associated with a common amyloidogenic intermediate, which may have been a relatively compact denatured conformation. We also suggest that the Handai amyloid burst inducer system will be useful for studying the mechanism of crystallization of proteins because proteins form crystals by the same mechanism as amyloid fibrils under supersaturation.

Measurement of amyloid formation by turbidity assay—seeing through the cloud

Detection of amyloid growth is commonly carried out by measurement of solution turbidity, a low-cost assay procedure based on the intrinsic light scattering properties of the protein aggregate. Here, we review the biophysical chemistry associated with the turbidimetric assay methodology, exploring the reviewed literature using a series of pedagogical kinetic simulations. In turn, these simulations are used to interrogate the literature concerned with in vitro drug screening and the assessment of amyloid aggregation mechanisms.

Ultrasonication-dependent formation and degradation of α-synuclein amyloid fibrils

Ultrasonication can be used to break the supersaturation of α-synuclein, a protein associated with Parkinsons disease, at pH7.4 above the critical concentration of fibrillation, thereby inducing the formation of amyloid fibrils. We speculated that ultrasonication could also be used to depolymerize preformed fibrils below the critical concentration. However, extensive ultrasonic irradiation transformed preformed fibrils into amorphous aggregates even above the critical concentration. Exposing preformed fibrils to the hydrophobic air-water interface of cavitation bubbles may have destabilized the fibrils and stabilized amorphous aggregates. Upon extensive ultrasonic irradiation, the accompanying decomposition of chemical structures was suggested when monitored by analytical ultracentrifugation. Amorphous aggregates produced by extensive ultrasonication showed higher cytotoxicity, suggesting that, although ultrasonication might be a useful approach for inactivating amyloid fibrils, potential cytotoxicity of amorphous aggregates should be considered.

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 Aβ 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.

Drastic acceleration of fibrillation of insulin by transient cavitation bubble

Amyloid-fibril formation of proteins can be accelerated by ultrasonic irradiation to the peptide solutions. Although this phenomenon contributes to understanding pathogenic behavior of amyloidosis, its physical mechanism has not been clarified, because several factors (cavitation, temperature increase, stirring effect, and so on) related to ultrasonic irradiation can participate in the fibrillation reaction. Here, we independently study contributions of the possible factors, using insulin, which is extremely stable and then suitable for the mechanism clarification. We find that the optimized ultrasonic irradiation can drastically accelerate the fibrillation reaction; the time for completing the reaction is shortened compared with the high-speed (1200rpm) stirring agitation by a factor of 430. The fibrillation reaction proceeds only when the subharmonic-mode intensity exceeds a threshold, indicating generation of the transient cavitation bubbles. Our results reveal that not the temperature increase but the transient cavitation bubbles work as the dominant accelerator of the fibrillation reaction.