PDF(Pubmed)
Abstract：
Conformational diseases, such as Alzheimer\'s, Parkinson\'s and Huntington\'s diseases as well as ataxias and fronto-temporal disorders, are part of common class of neurological disorders characterised by the aggregation and progressive accumulation of mutant proteins which display aberrant conformation. In particular, Huntington\'s disease (HD) is caused by mutations leading to an abnormal expansion in the polyglutamine (poly-Q) tract of the huntingtin protein (HTT), leading to the formation of inclusion bodies in neurons of affected patients. Furthermore, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of mutant HTT to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein. There is still no successful strategy to treat HD. In addition, the current understanding of the biological processes leading to the oligomerization and aggregation of proteins bearing the poly-Q tract has been derived from studies conducted on isolated poly-Q monomers and oligomers, whose structural properties are still unclear and often inconsistent. Here we describe a standardised biochemical approach to analyse by isopycnic ultracentrifugation the oligomerization of the N-terminal fragment of mutant HTT. The dynamic range of our method allows one to detect large and heterogeneous HTT complexes. Hence, it could be harnessed for the identification of novel molecular determinants responsible for the aggregation and the prion-like spreading properties of HTT in the context of HD. Equally, it provides a tool to test novel small molecules or bioactive compounds designed to inhibit the aggregation of mutant HTT.
摘要：
构象疾病,比如阿尔茨海默氏症，帕金森病和亨廷顿病以及共济失调和额颞部疾病，是常见的神经系统疾病的一部分，其特征是显示异常构象的突变蛋白的聚集和逐渐积累。特别是,亨廷顿病（HD）是由突变引起的，突变导致亨廷顿蛋白（HTT）的聚谷氨酰胺（poly-Q）束异常扩张，导致在受影响患者的神经元中形成包涵体。此外，最近的实验证据通过揭示突变HTT通过细胞外囊泡（EV）在细胞之间转移的能力，挑战了疾病的传统观点，允许突变蛋白接种涉及突变型和野生型蛋白的寡聚体。仍然没有成功的治疗HD的策略。此外,目前对导致蛋白质寡聚化和聚集的生物过程的理解携带poly-Q束已经从对分离的poly-Q单体和寡聚物进行的研究中得出，其结构性质尚不清楚，往往不一致。在这里，我们描述了一种标准化的生化方法，通过等密度超速离心分析突变HTT的N端片段的寡聚化。我们方法的动态范围允许检测大型和异质HTT复合物。因此，在HD的背景下，它可以用于鉴定导致HTT聚集和朊病毒样扩散特性的新型分子决定簇。同样,它提供了一种工具来测试旨在抑制突变HTT聚集的新型小分子或生物活性化合物。
