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

肉眼可见的包涵体含有洗涤剂不溶于洗涤剂的蛋白质纤维聚集物和扩张的聚谷氨酰胺(PolyQ)束，是多谷氨酰胺重复疾病的主要组织学特征，例如亨廷顿病和几个脊髓小脑性共济失调。多Q延伸完全由CAG密码子编码，这本质上很容易发生翻译移码。多聚Q重复序列中阅读框的移动将导致GCA编码的聚丙氨酸(PolA)或AGC编码的多丝氨酸(Polys)延伸，两者相比多Q重复序列具有更高的聚集倾向。在这里，我们采用广泛的体内和体外方法，旨在探索PolyQ伸展区域内的随机移码对疾病发病的影响，以及移码产物在调节毒性和重塑亲代PolyQ蛋白聚集途径中的作用。此外，我们将探讨依赖于细胞阶段或老化的环境因素(例如，同源谷氨酰tRNA的可用性)的变化是否会改变移码频率，从而改变多Q聚集模式。此外，对PolyQ病的细胞类型特异性的研究还将集中在延伸因子1A(EEF1A)上，并已被发现在病理性PolyQ聚集体中招募。EEF1A的两种细胞型特异性亚型(eEF1A1和eEF1A2)在很大程度上具有不重叠的表达模式：EF1A1几乎普遍表达，而EF1A2仅限于持久的有丝分裂后细胞。因此，我们试图了解eEF1A2的表达是否与增加的移码率有关，这反过来可以解释多聚Q介导的毒性的细胞类型特异性和有丝分裂后细胞中多聚Q疾病的主要表现。这一新的研究方向试图填补目前对聚集形成的分子事件与临床组织病理学关键特征(如细胞类型特异性、聚集过程的异质性和发病时间)之间联系的显著空白。这项建议是我们当前研究计划的合乎逻辑的扩展；它使我们能够建立我们在多聚Q蛋白聚集机制的结构研究方面的专业知识，并且我们已经建立了必要的实验程序，以在技术上实现这项建议的目标。

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.