Lysosomal Diseases Research Unit, South Australian Health and Medical Research Institute, North Terrace, Adelaide, South Australia 5000, Australia.
Lysosomal Diseases Research Unit, South Australian Health and Medical Research Institute, North Terrace, Adelaide, South Australia 5000, Australia.
Biochim Biophys Acta Proteins Proteom. 2017 Mar;1865(3):261-273. doi: 10.1016/j.bbapap.2016.12.008. Epub 2016 Dec 19.
Parkinson's disease (PD) presents with neuropathological inclusions called Lewy bodies, which are primarily composed of fibrillar α-synuclein. Recently, we characterized sheep with Gaucher disease and since GBA1 mutations represent the highest genetic risk factor for PD, we have investigated α-synuclein fibrillation in the sheep. Here we demonstrate that differences in six amino acid residues between sheep and human α-synuclein significantly alter in vitro fibril formation. Circular dichroism of recombinant human and sheep α-synuclein show that both proteins adopt the same secondary structure. Fibrils from human and sheep α-synuclein formed at pH7.0 or 4.5 were analyzed by Transmission Electron Microscopy (TEM). Unexpectedly, sheep α-synuclein form fibrils much less readily than human α-synuclein and this difference was more pronounced at the lysosomal pH of 4.5. Aggregation-propensity and intrinsic-solubility analysis revealed that sheep α-synuclein had lower aggregation-propensity and higher solubility. As a result of these observations, TEM was used to analyze fibrils formed at pH4.5 of various "sheep-like" human or "human-like" sheep mutant α-synucleins, together with their wild-type forms. Thioflavin T was used to monitor in situ α-synuclein fibril formation at pH7.0 and 4.5. Results show that "sheep-like" human α-synuclein has substantially lower fibril aggregation, and "human-like" sheep α-synuclein aggregates faster than wild-type forms, respectively. Seeding with WT human α-synuclein showed that "sheep-like" human α-synuclein could not be seeded, providing further evidence that sheep sequence is resistant to fibrillation. These findings provide new avenues to prevent/reduce fibrillation in PD, which may aid in the development of therapies.
帕金森病（PD）表现为称为路易体的神经病理学内含物，其主要由纤维状α-突触核蛋白组成。最近，我们对患有戈谢病的绵羊进行了特征描述，由于 GBA1 突变是 PD 的最高遗传风险因素，因此我们研究了绵羊中α-突触核蛋白的纤维形成。在这里，我们证明了绵羊和人类α-突触核蛋白之间六个氨基酸残基的差异显著改变了体外纤维形成。重组人类和绵羊α-突触核蛋白的圆二色性表明这两种蛋白质均采用相同的二级结构。在 pH7.0 或 4.5 下形成的人源和绵羊α-突触核蛋白纤维通过透射电子显微镜（TEM）进行分析。出乎意料的是，绵羊α-突触核蛋白形成纤维的速度远低于人类α-突触核蛋白，并且这种差异在溶酶体 pH4.5 时更为明显。聚集倾向和固有溶解度分析表明绵羊α-突触核蛋白的聚集倾向较低，溶解度较高。由于这些观察结果，TEM 用于分析在 pH4.5 下形成的各种“绵羊样”人或“人样”绵羊突变α-突触核蛋白及其野生型形式的纤维。噻唑黄素 T 用于监测 pH7.0 和 4.5 下原位α-突触核蛋白纤维形成。结果表明，“绵羊样”人α-突触核蛋白的纤维聚集程度明显较低，而“人样”绵羊α-突触核蛋白的聚集速度比野生型形式更快。用 WT 人α-突触核蛋白进行接种表明，“绵羊样”人α-突触核蛋白不能被接种，这进一步证明绵羊序列不易发生纤维形成。这些发现为预防/减少 PD 中的纤维形成提供了新的途径，这可能有助于开发治疗方法。
