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 Saccharomyces cerevisiae, 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 PTOH1 or inducible PCUP1 promoter. Using bacterial system for generation of extracellular amyloid aggregates C-DAG, we demonstrated that the N-terminal Toh1 fragment, containing amyloidogenic regions predicted in silico, 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 [PIN+] and [PSI+] 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）是一类由堆叠单体通过分子间β折叠稳定形成的无分支原纤维。部分淀粉样蛋白与不治之症密切相关，而另一类功能性淀粉样蛋白（functional amyloids）则参与调控多种重要生命过程。目前，学界对功能性淀粉样蛋白在野生生物中的分布普遍性与生物学意义仍缺乏深入了解。近年来，研究人员通过大规模蛋白质组筛选（large-scale proteome screening）这一新兴研究手段，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中。本研究证实，其中一种名为Toh1的蛋白具备典型淀粉样蛋白特性。在其自身启动子PTOH1或诱导型启动子PCUP1的驱动下表达时，Toh1-黄色荧光蛋白（YFP）融合蛋白可在酵母细胞内形成抗去污剂聚集复合物。研究团队利用可生成细胞外淀粉样聚集物的细菌表达系统C-DAG，开展了体外验证实验：包含计算机预测的淀粉样形成区域的Toh1 N端片段，可特异性结合刚果红染料；在正交偏光镜下观测时，该片段呈现出淀粉样蛋白特征性的"苹果绿"双折射现象；且通过透射电子显微镜（TEM）可清晰观察到其形成类淀粉样原纤维聚集结构。进一步实验表明，在携带[PIN+]与[PSI+]朊病毒（prions）的酵母细胞中，Toh1(20–365)-YFP融合蛋白的荧光聚集物与Rnq1C-青色荧光蛋白（CFP）、Sup35NM-青色荧光蛋白（CFP）的聚集物呈现高频共定位特征；同时，通过荧光共振能量转移（FRET）技术证实了这些聚集蛋白之间存在直接物理相互作用。这是目前已知的少数几例非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案例之一，该发现提示：不同淀粉样蛋白之间的相互作用，不仅可能取决于其一级结构的相似性，还可能与其二级结构及构象折叠的相似性密切相关。