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首页> 外文期刊>Biochemistry >Orthogonal cross-seeding: An approach to explore protein aggregates in living cells
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Orthogonal cross-seeding: An approach to explore protein aggregates in living cells

机译：正交交叉播种：探索活细胞中蛋白质聚集体的方法

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摘要

Protein aggregation is associated with the pathology of many diseases, especially neurodegenerative diseases. A variety of structurally polymorphic aggregates or preaggregates including amyloid fibrils is accessible to any aggregating protein. Preaggregates are now believed to be the toxic culprits in pathologies rather than mature aggregates. Although clearly valuable, understanding the mechanism of formation and the structural characteristics of these prefibrillar species is currently lacking. We report here a simple new approach to map the nature of the aggregate core of transient aggregated species directly in the cell. The method is conceptually based on the highly discriminating ability of aggregates to recruit new monomeric species with equivalent molecular structure. Different soluble segments comprising parts of an amyloidogenic protein were transiently pulse-expressed in a tightly controlled, time-dependent manner along with the parent aggregating full-length protein, and their recruitment into the insoluble aggregate was monitored immunochemically. We used this approach to determine the nature of the aggregate core of the metastable aggregate species formed during the course of aggregation of a chimera containing a long polyglutamine repeat tract in a bacterial host. Strikingly, we found that different segments of the full-length protein dominated the aggregate core at different times during the course of aggregation. In its simplicity, the approach is also potentially amenable to screen also for compounds that can reshape the aggregate core and induce the formation of alternative nonamyloidogenic species.
机译：蛋白质聚集与许多疾病，特别是神经退行性疾病的病理有关。各种结构多态性聚集体或预聚集体，包括淀粉样原纤维，都可以被任何聚集蛋白所利用。现在认为预聚集体是病理中的毒菌，而不是成熟的聚集体。尽管很有价值，但目前尚缺乏对这些原纤维种的形成机理和结构特征的了解。我们在这里报告了一种简单的新方法，可以直接在细胞中映射瞬时聚集物种的聚集核的性质。从概念上讲，该方法基于聚集体对具有相同分子结构的新单体种类的高度区分能力。以紧密控制的，时间依赖性的方式与组成全长蛋白质的亲本一起，瞬时地表达包括部分淀粉样蛋白的蛋白质的不同可溶性片段，并通过免疫化学方法监测它们募集到不溶性聚集物中。我们使用这种方法来确定在细菌宿主中包含长聚谷氨酰胺重复序列的嵌合体聚集过程中形成的亚稳态聚集物种的聚集核的性质。令人惊讶的是，我们发现全长蛋白质的不同片段在聚集过程中的不同时间主导着聚集核心。简单来说，该方法也可能适合筛选也可以重塑聚集核并诱导形成非淀粉样物质的化合物。

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