The impact of the E46K mutation on the properties of α-synuclein in its monomeric and oligomeric states
The impact of the E46K mutation on the properties of α-synuclein in its monomeric and oligomeric states
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DOI：
10.1021/bi7000246
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发表时间：
2007-06-19
期刊：
影响因子：
2.9
通讯作者：
Lansbury, Peter T., Jr.
中科院分区：
文献类型：
--
作者：
Fredenburg, Ross A.;Rospigliosi, Carla;Lansbury, Peter T., Jr.
待鉴定的神经元蛋白α-突触核蛋白的第三种和最近鉴定的帕金森病相关变体（E46 K）导致广泛的脑病理学和早发性帕金森症状（Zarranz等人（2004）Ann.Neurol.2005）。55，164-173）。在此，我们提出了E46 K α-突触核蛋白在各种聚集状态下的生物化学和生物物理学特征。圆二色性和核磁共振光谱表明，E46 K突变的结果在单体蛋白质的构象的微妙变化，无论是在溶液中的自由和在SDS胶束的存在下。然而，它不改变磷脂存在下蛋白质的整体螺旋倾向。E46 K α-突触核蛋白在体外形成不溶性纤维的速度比野生型蛋白更快，电子显微镜显示，E46 K α-突触核蛋白纤维具有典型的淀粉样蛋白超微结构。E46 K α-突触核蛋白原纤维，可溶性聚集体，在从单体形式的α-突触核蛋白的纤维状形式的过渡过程中形成，其特征在于通过电子显微镜和凝胶过滤，并发现包括环形物种。E46 K和野生型α-突触核蛋白原纤维的亚组分的独特能力，含有孔样物质透化脂质囊泡在体外使用实时色谱法证明。与简单化的预期相反，E46 K突变降低了原纤维组分中每摩尔蛋白质的原纤维总量和透化活性量。这些结果表明，如果α-突触核蛋白的孔样活性对于神经毒性很重要，那么神经元细胞质中一定存在一些因素，可以逆转体外观察到的E46 K与WT α-突触核蛋白内在特性的趋势。
The third and most recently identified Parkinson's disease-linked variant of the neuronal protein alpha-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164-173). Herein, we present biochemical and biophysical characterization of E46K alpha-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K alpha-synuclein formed insoluble fibrils in vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K alpha-synuclein fibrils possess a typical amyloid ultrastructure. E46K alpha-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of alpha-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type alpha-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of alpha-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT alpha-synuclein that are observed in vitro.