Molecular Misreading: The Frameshift Species as Modulating Agents of Aggregation and Neurodegenerative Phenotype of Polyglutamine Proteins

肉眼可见的包涵体是多聚谷氨酰胺重复疾病的主要组织学标志，它含有不溶于洗涤剂的纤维状蛋白聚集体，并具有扩展的多聚谷氨酰胺（polyQ）道。例如，在一个实施例中，亨廷顿氏病和几种脊髓小脑共济失调。polyQ片段仅由CAG密码子编码，其本质上高度倾向于翻译移码。polyQ链段内阅读框的移位将导致GCA编码的聚丙氨酸（polyA）或AGC编码的聚丝氨酸（polyS）链段，这两者与polyQ重复序列相比具有更高的聚集倾向。在这里，使用广泛的体内和离体方法，我们的目标是探索polyQ延伸内的随机移码对疾病发作的影响，以及移码产物在调节毒性和重塑亲本polyQ蛋白的聚集途径中的作用。此外，我们将讨论环境因素的变化（例如，依赖于细胞阶段或老化的同源的氨酰-tRNA可用性）可以改变移码频率，从而改变polyQ聚集模式。关于polyQ疾病中细胞类型特异性的研究将另外关注延伸因子1A（eEF 1A），并且已发现其在病理性polyQ聚集体中被募集。eEF 1A的两种细胞类型特异性亚型（eEF 1A 1和eEF 1A 2）具有很大程度上不重叠的表达模式：而EF 1A 1几乎无处不在地表达，EF 1A 2仅限于持久的有丝分裂后细胞。因此，我们试图了解eEF 1A 2的表达是否与增加的移码率有关，这反过来可以解释polyQ的细胞类型特异性，介导的毒性和有丝分裂后细胞中polyQ疾病的主要表现。我们研究的这一新方向旨在填补目前对聚集体形成的分子事件与临床主要特征之间联系的理解中的显着空白。组织病理学（例如，细胞类型特异性、聚集过程的异质性和发作时间）。该提案是我们当前研究计划的逻辑延伸;它使我们能够建立我们对polyQ蛋白聚集机制结构研究的专业知识，并且我们已经建立了必要的实验程序，以在技术上实现该提案的目标。

Macroscopically discernible inclusions, containing detergent-insoluble fibrillar aggregates of proteins with expanded polyglutamine (polyQ) tract are the major histological hallmark of the polyglutamine-repeat diseases, e. g., Huntington’s disease and several spinocerebellar ataxias. The polyQ stretches are exclusively encoded by the CAG codon, which is intrinsically highly prone to translational frameshifting. A shift of the reading frame within the polyQ stretch will lead to GCA encoded polyalanine (polyA) or AGC encoded polyserine (polyS) stretches, both of which compared the polyQ repeats posses a higher tendency to aggregate. Here, using a broad spectrum of in vivo and ex vivo approaches we aim to explore the impact of the stochastic frameshifting within the polyQ stretches on the disease onset and the role of frameshift products in modulating the toxicity and re-shaping the aggregation pathway of the parental polyQ protein. Furthermore, we will address whether changes in the environmental factors (e.g., cognate glutaminyl-tRNA availability) dependent on the cell stage or aging can alter frameshifting frequency and thus the polyQ aggregation pattern. The research on the cell-type specificity in polyQ diseases will additionally focus on elongation factor 1A (eEF1A) and has been found to be recruited in pathologic polyQ-aggregates. The two cell-type specific isoforms of eEF1A (eEF1A1 and eEF1A2) possess largely non-overlapping expression patterns: whereas EF1A1 is nearly ubiquitously expressed, EF1A2 is restricted to long lasting postmitotic cells. Thereby, we seek to understand whether expression of eEF1A2 can be related to an increased frameshift rate, which in turn could explain the cell-type specificity of polyQ-mediated toxicity and the predominant manifestation of polyQ diseases in postmitotic cells.This new direction of our research seeks to fill the remarkable void in current understanding of the link between the molecular events of aggregate formation and key features of the clinical histopathology (e.g., cell-type specificity, heterogeneity of the aggregation process and time of onset). This proposal is a logical extension of our current research program; it allows us to build on our expertise on structural studies on the aggregation mechanism of polyQ proteins, and we have established the experimental procedures necessary to technically accomplish the goals of this proposal.