使用双分子荧光互补测定法监控α-突触核蛋白的低聚和聚集：您所看到的并不总是所得到的,Journal of Neurochemistry

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使用双分子荧光互补测定法监控α-突触核蛋白的低聚和聚集：您所看到的并不总是所得到的
Journal of Neurochemistry ( IF 4.2 ) Pub Date : 2020-08-08 , DOI: 10.1111/jnc.15147
Bryan Frey ₁ , Abdelrahman AlOkda ₁ , Matthew P Jackson ₂ , Nathan Riguet ₁ , James A Duce ₂ , Hilal A Lashuel ₁

Affiliation

双分子荧光互补（BiFC）是十年前引入的一种方法，用于监测完整细胞中的α-突触核蛋白（α-syn）寡聚。从那时起，基于荧光信号的增加与增加的α-syn寡聚或聚集相关的假设，开发了几种α-synBiFC细胞分析和动物模型。尽管在机械研究，靶标验证和药物筛选中越来越多地使用这些测定法和模型，但尚未有报道（1）在生物化学水平上验证BiFC荧光信号与α-syn寡聚相关的程度；（2）提供了由BiFC形成的低聚物和聚集体的结构表征。为了弥补这一知识鸿沟，我们首先使用多种生化方法和技术分析了来自三个不同实验室的HEK-293和SH-SY5Y细胞中最常用的BiFC系统之一α-syn-Venus各个成分的表达水平和低聚特性。接下来，我们研究了两个BiFC片段共表达时α-syn的生化特性和聚集特性。我们的结果表明（1）与α-syn（Vn-α- syn）; （2）Vn-α-syn具有形成低聚物和高阶聚集体的倾向。（3）一个或两个片段的表达不会导致α-syn原纤维或细胞内含物的形成。此外，我们的结果表明，只有少量的Vn-α-syn参与了荧光BiFC络合物的形成，并且某些荧光信号可能是由Vn-α-syn中的α-syn-Vc的缔合或截留引起的。聚集体。N末端片段主要以聚集状态存在这一事实也表明，在使用该系统研究细胞或体内α-syn寡聚时，必须谨慎行事。总之，我们的结果表明，应基于生化水平对基于α-synBiFC系统的寡聚化，聚集和细胞间传播的细胞和动物模型进行全面表征，以确保它们重现所关注的过程并对其进行测量。用于测量。

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Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: What you see is not always what you get

Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that (1) validate the extent to which the BiFC fluorescent signal correlates with α-syn oligomerization at the biochemical level; (2) provide a structural characterization of the oligomers and aggregates formed by the BiFC. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of α-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH-SY5Y cells from three different laboratories using multiple biochemical approaches and techniques. Next, we investigated the biochemical and aggregation properties of α-syn upon co-expression of both BiFC fragments. Our results show that (1) the C-terminal-Venus fused to α-syn (α-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to α-syn (Vn-α-syn); (2) Vn-α-syn exhibits a high propensity to form oligomers and higher-order aggregates; and (3) the expression of either or both fragments does not result in the formation of α-syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn-α-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of α-syn-Vc in Vn-α-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate α-syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission based on the α-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.

更新日期：2020-08-08

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