Aggregation Mechanisms of PolyQ-containing Proteins: Structure and Cytotoxicity of the Metastable Intermediate Species

不溶性纤维聚集体是多聚谷氨酰胺（polyQ）-重复疾病的主要组织病理学特征，但其在疾病病理学中的作用仍有争议。以下三个假设可以解释文献中报道的毒性和体内聚集体形成之间的相关性差：1）具有不同毒性的多种亚稳态物质的顺序组装途径，2）具有不同稳定性和毒性的各种瞬时物质的多种竞争性聚集途径，以及3）各种内部（例如，polyQ-侧翼序列）和分子间因子（例如，老化、应激反应）可改变聚集途径。为了验证这些假设，我们提出了新的体内实验，允许直接映射结构（例如，涉及聚集体核心、柔性区的序列）和在具有扩展的多聚谷氨酰胺片段的亨廷顿蛋白（Htt）的外显子1编码片段的聚集途径中出现的聚集体种类的特征（开启或关闭途径）。此外，使用广泛的体内和体外方法，我们的目标是确定亚稳态聚集体的结构和分子相互作用的聚集体形态，它们在细胞环境中的传播和它们的毒性。 结构和细胞毒性之间的联系将是我们进一步的主要目标的机制基础：制定策略，以不利于有毒物质的形成。因此，主要重点将是探索自然进化的机制，即细胞补偿反应，以及不同类型的渗透剂如何重塑polyQ蛋白的聚集途径，并促进非淀粉样蛋白生成，无毒的关闭途径物质的形成。

Insoluble fibrillar aggregates are the major histophological feature of polyglutamine (polyQ)-repeat diseases however their role in the disease pathology is still controversial. Following three hypotheses could explain the reported in the literature poor correlation between toxicity and aggregate formation in vivo: 1) a sequential assembly pathway with multiple metastable species with different toxicity, 2) multiple competing aggregation pathways with various transient species with different stability and toxicity, and 3) various intra- (e.g., polyQ-flanking sequences) and intermolecular factors (e.g., aging, stress response) could alter the aggregation pathway(s). To test these hypotheses, we propose new in vivo experiments allowing to directly map the structure (e.g., sequences involved in the aggregate core, flexible regions) and the character (on- or off- pathway) of the aggregate species emerging in the aggregation pathway of the exon 1 encoded fragment of huntingtin (Htt) with expanded polyglutamine stretch. In addition, using a broad spectrum of in vivo and ex vivo approaches we aim to determine the structure of the metastable aggregates and the molecular interactions governing aggregate morphology, their propagation in the cellular context and their toxic properties. The link between the structure and cytotoxicity will be the mechanistic foundation for our further major goal: to develop strategies to disfavor the formation of the toxic species. Thereby, the main focus will be to explore a naturally evolved mechanism, the osmocompensatory response, and how different types of osmolytes can re-shape the aggregation pathway of polyQ proteins and promote the formation of non-amyloidogenic, non-toxic off-pathway species.