Division of Molecular Structure, Medical Research Council, National Institute for Medical Research, London, United Kingdom.
Biophys J. 2011 Apr 20;100(8):2033-42. doi: 10.1016/j.bpj.2011.02.056.
Fibrillar aggregation of the protein ataxin-3 is linked to the inherited neurodegenerative disorder Spinocerebellar ataxia type 3, a member of the polyQ expansion disease family. We previously reported that aggregation and stability of the nonpathological form of ataxin-3, carrying an unexpanded polyQ tract, are modulated by its N-terminal Josephin domain. It was also shown that expanded ataxin-3 aggregates via a two-stage mechanism initially involving Josephin self-association, followed by a polyQ-dependent step. Despite this recent progress, however, the exact mechanism of ataxin-3 fibrilization remains elusive. Here, we have used electron microscopy, atomic force microscopy, and other biophysical techniques to characterize the morphological and mechanical properties of nonexpanded ataxin-3 fibrils. By comparing aggregates of ataxin-3 and of the isolated Josephin domain, we show that the two proteins self-assemble into fibrils with markedly similar features over the temperature range 37-50°C. Estimates of persistence length and Young's modulus of the fibrils reveal a great flexibility. Our data indicate that, under physiological conditions, during early aggregation Josephin retains a nativelike secondary structure but loses its enzymatic activity. The results suggest a key role of Josephin in ataxin-3 fibrillar aggregation.
蛋白 ataxin-3 的纤维状聚集与遗传性神经退行性疾病脊髓小脑共济失调 3 型有关，后者是多聚谷氨酰胺扩展疾病家族的一员。我们之前曾报道过，非病理性形式的 ataxin-3，其 polyQ 片段未扩展，其 N 端 Josephin 结构域可调节其聚集和稳定性。还表明，扩展的 ataxin-3 通过两阶段机制聚集，最初涉及 Josephin 自身缔合，然后是依赖 polyQ 的步骤。然而，尽管取得了这一最新进展，但 ataxin-3 纤维化的确切机制仍不清楚。在这里，我们使用电子显微镜、原子力显微镜和其他生物物理技术来表征非扩展 ataxin-3 原纤维的形态和机械特性。通过比较 ataxin-3 和分离的 Josephin 结构域的聚集体，我们表明在 37-50°C 的温度范围内，两种蛋白质均可自组装成具有明显相似特征的原纤维。原纤维的持久长度和杨氏模量的估计表明其具有很大的柔韧性。我们的数据表明，在生理条件下，在早期聚集过程中，Josephin 保留了天然的二级结构，但失去了其酶活性。这些结果表明 Josephin 在 ataxin-3 纤维状聚集中起关键作用。
