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首页> 外文期刊>Journal of Molecular Biology >Rapid Self-assembly of alpha-Synuclein Observed by In Situ Atomic Force Microscopy.
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Rapid Self-assembly of alpha-Synuclein Observed by In Situ Atomic Force Microscopy.

机译：原位原子力显微镜观察到的α-突触核蛋白的快速自组装。

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Self-assembly of alpha-synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson's disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy to continuously monitor the self-assembly of wild-type alpha-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant alpha-synuclein(1-108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(+/-0.2)nm in height and presumably representing individual beta-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite substrates, suggesting that negatively charged surfaces promote alpha-synuclein self-assembly. In the presence of spermidine or spermine 5.9(+/-1.0)nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. alpha-Synuclein spheroid formation depended critically on polyamine binding to the C terminus, revealing a promoting effect of the C terminus on alpha-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of alpha-synuclein self-assembly and identifies key factors modulating the aggregation process.
机译：α-突触核蛋白的自组装导致多种形态的蛋白质聚集体，与帕金森氏病和其他被称为突触核蛋白病的神经退行性疾病的发病机理有关。除了其生物医学相关性外，这种聚集过程还代表了未折叠蛋白相互转化为具有典型淀粉样蛋白特征的纳米结构的过程。我们使用原位敲击模式原子力显微镜连续监测野生型α-突触核蛋白，其疾病相关突变体A30P和A53T以及C端截短的变体α-突触核蛋白（1-108）的自组装。根据实验条件，即pH，蛋白质浓度，多胺浓度，温度和支持底物，观察到不同的聚集模式。在pH值为7.5的情况下，在几分钟内在云母基质上形成了不存在生物多胺亚精胺或亚精胺的，高度为1.1（+/- 0.2）nm且可能代表单个β-片状结构的细长片。它们的取向由基底的结晶亚结构指示。相反，在疏水性高度取向的热解石墨基底上未观察到片状形成，这表明带负电荷的表面促进了α-突触核蛋白的自组装。在亚精胺或亚精胺的存在下，优先形成5.9（+/- 1.0）nm的高球状结构，与以前报道的几种淀粉样蛋白的结构相似，并具有神经毒性作用。 α-突触核蛋白球体的形成主要取决于多胺与C末端的结合，揭示了C末端对结合状态的α-突触核蛋白组装的促进作用。在极少数情况下，观察到从原球或预先形成的聚集体中产生原纤维。在pH 5.0时，最初形成原纤维，并在聚集过程中掺入无定形聚集体中，为淀粉样原纤维表面在无定形聚集中充当成核位点提供了证据。这项研究提供了对α-突触核蛋白自组装的不同模式的直接见解，并确定了调节聚集过程的关键因素。

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