From the Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
the ELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Eötvös Loránd University, Budapest 1117, Hungary, and.
J Biol Chem. 2018 Sep 21;293(38):14775-14785. doi: 10.1074/jbc.RA118.004683. Epub 2018 Aug 3.
Several serious diseases are associated with crystal-like amyloid fibrils or glass-like amorphous aggregates of denatured proteins. However, protein aggregation involving both types of aggregates has not yet been elucidated in much detail. Using a protein associated with dialysis-related amyloidosis, β-microglobulin (β2m), we previously demonstrated that amyloid fibrils and amorphous aggregates form competitively depending on salt (NaCl) concentration. To examine the generality of the underlying competitive mechanisms, we herein investigated the effects of heat on acid-denatured β2m at pH 2. Using thioflavin fluorescence, CD, and light scattering analysis along with atomic force microscopy imaging, we found that the temperature-dependent aggregation of β2m markedly depends on NaCl concentration. Stepwise transitions from monomers to amyloids and then back to monomers were observed at low NaCl concentrations. Amorphous aggregates formed rapidly at ambient temperatures at high NaCl concentrations, but the transition from amorphous aggregates to amyloids occurred only as the temperature increased. Combining the data from the temperature- and NaCl-dependent transitions, we constructed a unified phase diagram of conformational states, indicating a parabolic solubility curve with a minimum NaCl concentration at ambient temperatures. Although amyloid fibrils formed above this solubility boundary, amorphous aggregates dominated in regions distant from this boundary. Kinetic competition between supersaturation-limited slow amyloid fibrillation and supersaturation-unlimited fast amorphous aggregation deformed the phase diagram, with amyloid regions disappearing with fast titration rates. We conclude that phase diagrams combining thermodynamics and kinetics data provide a comprehensive view of β2m aggregation exhibiting severe hysteresis depending on the heat- or salt-titration rates.
几种严重的疾病与类似晶体的淀粉样原纤维或变性蛋白质的玻璃状无定形聚集物有关。然而，涉及这两种聚集物的蛋白质聚集尚未得到详细阐明。使用与透析相关淀粉样变性相关的蛋白质β-微球蛋白（β2m），我们之前证明了淀粉样原纤维和无定形聚集物根据盐（NaCl）浓度形成竞争。为了研究潜在竞争机制的普遍性，我们在此研究了热对 pH 2 下酸变性β2m 的影响。通过使用硫黄素荧光、CD 和光散射分析以及原子力显微镜成像，我们发现β2m 的温度依赖性聚集显著依赖于 NaCl 浓度。在低 NaCl 浓度下观察到从单体到淀粉样物再到单体的逐步转变。在高 NaCl 浓度下，在环境温度下迅速形成无定形聚集物，但只有当温度升高时，才会从无定形聚集物转变为淀粉样物。将温度和 NaCl 依赖性转变的数据结合起来，我们构建了构象状态的统一相图，表明在环境温度下具有最小 NaCl 浓度的抛物线溶解度曲线。虽然淀粉样原纤维在该溶解度边界之上形成，但无定形聚集物在远离该边界的区域占主导地位。过饱和度限制的缓慢淀粉样纤维形成和过饱和度不受限制的快速无定形聚集之间的动力学竞争使相图变形，随着快速滴定速率，淀粉样区域消失。我们得出的结论是，结合热力学和动力学数据的相图提供了一个综合视图，显示了β2m 聚集表现出严重的滞后现象，这取决于加热或盐滴定速率。
