Department of Biochemistry, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan.
Research and Development, ULVAC, Chigasaki, Kanagawa, Japan.
PLoS One. 2018 Oct 5;13(10):e0205090. doi: 10.1371/journal.pone.0205090. eCollection 2018.
Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.
在某些条件下，淀粉样纤维可以通过自组装过程形成三维网络并形成水凝胶。当抗氧化酶铜/锌超氧化物歧化酶（SOD1）发生错误折叠时，会形成纤维状聚集体，这是某种家族性肌萎缩侧索硬化症（ALS）的标志。然而，SOD1 纤维是否可以组装成水凝胶仍有待检验。在这里，我们表明 SOD1 多肽在 pH 3.0 和 4.0 下发生水凝胶化，同时形成硫黄素 T 阳性纤维，而在 pH 5.0 下则形成沉淀。粘弹性分析的结果表明，SOD1 水凝胶（2%）的性质与 0.25%琼脂糖凝胶相似，略脆弱。此外，通过石英晶体微天平介电分析监测表明，在 pH 3.0 和 4.0 下水凝胶化条件下固定在传感器上的 SOD1 变性会导致有效声厚从约 3.3nm（折叠刚性形式）分别增加到约 50nm 和约 100nm（伸展富水状态）。相比之下，当 SOD1 在相同条件下在 pH 5.0 下变性时，形成具有约 10nm 有效声厚的紧密贫水状态。向 pH 4.0 样品中添加生理浓度的 NaCl 会诱导 SOD1 进一步伸展并结合更多水分子（有效声厚约 200nm），但抑制水凝胶形成。这些结果表明，在自组装之前，蛋白质不同的变性中间状态在决定聚集物的特性方面起着主要作用，并且在球状蛋白的情况下，在分子间组装之前和/或期间需要向合适水平的伸展富水中间状态进行构象变化，以进行纤维化和水凝胶化。
