Amyloid fibrils are fibrillar and ordered aggregates of denatured proteins, associated with Alzheimer’s disease and Parkinson’s disease (PD) and dialysis-related amyloidosis (DRA). Today, more than 40 human amyloidogenic proteins and peptides are known. We have investigated mechanism of amyloid formation from the viewpoint of supersaturation- and solubility-limited protein phase transition, and have developed an instrument, HANABI, which can promote amyloid formation of proteins by ultrasonication and enable the rapid analysis.
At first, to reveal the mechanism of amyloid formation, we examined the effects of negatively charged biomolecules such as polyphosphate (polyP) and ATP on amyloid formation of β2-microblobulin (β2m), the protein responsible for DRA, and α-synuclein (αSyn), the protein responsible for PD, under ultrasonication using HANABI. As a result, polyP remarkably accelerated amyloid formation of both β2m and αSyn by the counter anion-binding and the preferential hydration at relatively lower and higher concentrations of polyP, respectively, in the concentration-dependent distinct manners. These bimodal concentration-dependent effects were observed in ATP- and salts-induced amyloid formation. On the other hands, macromolecular crowding environment within the LLPS also promoted amyloid formation of aSyn at surface of droplets through the depletion effect.
Although these interactions are like those stabilizing the native state, they simultaneously promote amyloid formation through intermolecular interactions upon breaking supersaturation where the concentration of amyloidogenic protein is above thermodynamic solubility. HANABI system was also used to gain insights into the pathogenesis of DRA with a patient’s sera in the context of proteostasis and molecular crowding. Further studies on the mechanism of amyloid formation using HANABI will contribute to developing therapeutic strategies against amyloidosis as well as understanding the biological significance of supersaturation on the amyloidome.