Amyloid properties of the yeast cell wall protein Toh1 and its interaction with prion proteins Rnq1 and Sup35

Amyloids are non-branching fibrils that are composed of stacked monomers stabilized by intermolecular β-sheets. Some amyloids are associated with incurable diseases, whereas others, functional amyloids, regulate different vital processes. The prevalence and significance of functional amyloids in wildlife are still poorly understood. In recent years, by applying new approach of large-scale proteome screening, a number of novel candidate amyloids were identified in the yeast <i>Saccharomyces cerevisiae</i>, many of which are localized in the yeast cell wall. In this work, we showed that one of these proteins, Toh1, possess amyloid properties. The Toh1-YFP hybrid protein forms detergent-resistant aggregates in the yeast cells while being expressed under its own <i>P_TOH1</i> or inducible <i>P_CUP1</i> promoter. Using bacterial system for generation of extracellular amyloid aggregates C-DAG, we demonstrated that the N-terminal Toh1 fragment, containing amyloidogenic regions predicted <i>in silico</i>, binds Congo Red dye, manifests ‘apple-green’ birefringence when examined between crossed polarizers, and forms amyloid-like fibrillar aggregates visualized by TEM. We have established that the Toh1(20–365)-YFP hybrid protein fluorescent aggregates are co-localized with a high frequency with Rnq1C-CFP and Sup35NM-CFP aggregates in the yeast cells containing [<i>PIN⁺</i>] and [<i>PSI⁺</i>] prions, and physical interaction of these aggregated proteins was confirmed by FRET. This is one of a few known cases of physical interaction of non-Q/N-rich amyloid-like protein and Q/N-rich amyloids, suggesting that interaction of different amyloid proteins may be determined not only by similarity of their primary structures but also by similarity of their secondary structures and of conformational folds.

淀粉样蛋白（Amyloids）是由经分子间β折叠稳定的堆叠单体构成的无分支原纤维。部分淀粉样蛋白与不治之症相关，而另一类功能性淀粉样蛋白（functional amyloids）则调控多种生命活动。目前学界对功能性淀粉样蛋白在野生生物中的普遍性与重要性仍缺乏深入认知。近年来，通过应用大规模蛋白质组筛选的新方法，研究人员在酿酒酵母（Saccharomyces cerevisiae）中鉴定出多个新型候选淀粉样蛋白，其中多数定位于酵母细胞壁。本研究证实，其中一种蛋白Toh1具备淀粉样蛋白特性。在自身P_TOH1启动子或可诱导型P_CUP1启动子的驱动下表达时，Toh1-YFP融合蛋白可在酵母细胞内形成抗去污剂聚集物。利用可生成胞外淀粉样聚集物C-DAG的细菌表达系统，研究人员证明：包含经计算机预测（in silico）得到的淀粉样生成区域的Toh1 N端片段可结合刚果红染料，在正交偏光镜下呈现“苹果绿”双折射现象，并形成经透射电子显微镜（TEM）可视化的类淀粉样纤维状聚集物。本研究还发现，在携带[PIN+]和[PSI+]朊病毒的酵母细胞中，Toh1(20–365)-YFP融合蛋白的荧光聚集物与Rnq1C-CFP、Sup35NM-CFP聚集物存在高频共定位，且通过荧光共振能量转移（FRET）证实了这些聚集蛋白间存在物理相互作用。这是已知的非Q/N富集型类淀粉样蛋白与Q/N富集型淀粉样蛋白发生物理相互作用的少数案例之一，该结果提示：不同淀粉样蛋白间的相互作用不仅可能由其一级结构的相似性决定，也可能与其二级结构及构象折叠的相似性相关。