Aggregation-phase diagrams of β2-microglobulin reveal temperature and salt effects on competitive formation of amyloids versus amorphous aggregates
Aggregation-phase diagrams of β2-microglobulin reveal temperature and salt effects on competitive formation of amyloids versus amorphous aggregates
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DOI：
10.1074/jbc.ra118.004683
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发表时间：
2018-09-21
影响因子：
4.8
通讯作者：
Goto, Yuji
中科院分区：
文献类型：
--
作者：
Adachi, Masayuki;Noji, Masahiro;Goto, Yuji
一些严重的疾病与晶体样淀粉样原纤维或变性蛋白质的玻璃样无定形聚集体有关。然而，涉及这两种聚集体的蛋白质聚集尚未被详细阐明。利用与透析相关的淀粉样变性相关的蛋白β(2)-微球蛋白（β 2m），我们先前证明淀粉样原纤维和无定形聚集体的形成取决于盐（NaCl）浓度。为了研究潜在竞争机制的普遍性，我们在此研究了pH为2时热对酸变性β 2m的影响。利用硫黄素荧光、CD和光散射分析以及原子力显微镜成像，我们发现β 2m的温度依赖性聚集明显依赖于NaCl浓度。在低NaCl浓度下，观察到从单体到淀粉样蛋白再到单体的逐步转变。在高NaCl浓度的环境温度下，无定形聚集体迅速形成，但从无定形聚集体到淀粉样蛋白的转变只有在温度升高时才会发生。结合温度和NaCl依赖性转变的数据，我们构建了一个统一的构象态相图，表明在环境温度下，NaCl浓度最低时，其溶解度呈抛物线曲线。尽管淀粉样蛋白原纤维在这一溶解度边界以上形成，但在远离这一边界的区域，无定形聚集体占主导地位。过饱和受限的慢淀粉样蛋白纤颤和过饱和无限制的快速非晶态聚集之间的动力学竞争使相图变形，淀粉样蛋白区域随着滴定速率的加快而消失。我们得出结论，结合热力学和动力学数据的相图提供了β 2m聚集的全面视图，显示出严重的滞后，这取决于热滴定率或盐滴定率。
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, beta(2)-microglobulin (beta 2m), 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 beta 2m 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 beta 2m 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 beta 2m aggregation exhibiting severe hysteresis depending on the heat- or salt-titration rates.