ROLE OF CHAIN LENGTH AND SEQUENCE CONTEXTS ON POLYGLUTAMINE OLIGOMERIZATION

 描述(申请人提供)：亨廷顿病(HD)是一种毁灭性的神经退行性疾病，由亨廷顿蛋白(Htt)基因外显子1的CAG密码子突变扩展引起。CAG重复序列的扩展导致亨廷顿蛋白N-末端区域的聚谷氨酰胺结构域扩大。Htt的N-末端片段(Htt-NTF)是通过全长蛋白的蛋白分解或突变的mRNA转录产物的异常剪接而产生的。这些碎片最终在中型棘神经元和小脑神经元内形成不溶的核内包涵体。HTT-NTF包括17个残基的N-末端延伸(N17)、多Q结构域和38个残基的富含Pro的C-末端延伸(C38)。我们已经表明，N17拉伸加速了纤维的形成，而C38拉伸破坏了不溶性纤维的稳定，并稳定了大的可溶性球形聚集体。最近在转基因小鼠中的研究表明，N17基因的缺失会导致极端的神经毒性和迅速的神经功能衰退。这些特征是HD发病和发展的典型特征。体内研究结合我们最近的发现，提出了由C38稳定的可溶性球形聚集体可能是HD神经毒性的来源的建议。在这一竞争性更新应用中，我们将寻求一种独特的方法，将新颖的多尺度计算机模拟与体外生物物理实验相结合，以促进我们对Htt-NTF的聚集机制和相行为的理解。我们的研究得到了我们新的粗粒度模拟方法的帮助。这些模拟结合新颖的体外实验将帮助我们了解C38模块如何稳定可溶的球形聚集体，同时减缓缺乏N17模块的Htt-NTF中从可溶球体到不溶纤维的转化。我们还试图了解N17模块的纤维稳定作用与C38的纤维破坏作用是如何竞争的，这些作用跨越具有不同长度的多Q链的Htt-NTF的外显子1。清楚地了解多聚Q长度、侧翼序列模块N17和C38之间的相互作用所形成的聚集机制和结构对于辨别Htt-NTF聚集和神经毒性之间的联系是绝对必要的。

 DESCRIPTION (provided by applicant): Huntington's disease (HD) is a devastating neurodegenerative disease that is caused by mutational expansion of the CAG codon in exon 1 of the huntingtin (Htt) gene. The CAG repeat expansion leads to an expanded polyglutamine domain in the N-terminal region of the huntingtin protein. N-terminal fragments of Htt (Htt-NTFs) are generated by proteolysis of the full-length protein or by aberrant splicing of mutant mRNA transcripts. These fragments end up as insoluble intranuclear inclusions within medium spiny and cerebellar neurons. Htt-NTFs encompass a 17-residue N-terminal stretch (N17), a polyQ domain, and a 38-residue proline-rich C-terminal stretch (C38). We have shown that the N17 stretch accelerates fibril formation whereas the C38 stretch destabilizes insoluble fibrils and stabilizes large soluble spherical aggregates. Recent studies in transgenic mice show that the deletion of N17 leads to extreme neurotoxicity and rapid neurological decline. These features typify the onset and progression of HD. The in vivo studies taken together with our recent findings, lead to the proposal that soluble spherical aggregates, which are stabilized by C38, might be the source of neurotoxicity in HD. In this competitive renewal application, we will pursue a unique approach that combines novel multiscale computer simulations with in vitro biophysical experiments to advance our understanding of the aggregation mechanisms and phase behavior of Htt-NTFs. Our studies are aided by our new methodologies for coarse-grained simulations. These simulations combined with novel in vitro experiments will help us understand how the C38 module stabilizes soluble spherical aggregates while slowing the conversion from soluble spheres to insoluble fibrils in Htt-NTFs that lack the N17 module. We also seek to understand how the fibril stabilizing effect of the N17 module competes with the fibril destabilizing effects of C38 in exon 1 spanning Htt-NTFs with polyQ tracts of different lengths. A clear understanding of the aggregation mechanisms and the structures that form as the result of the interplay among polyQ length, the flanking sequence modules N17 and C38 is absolutely necessary to discern the connection between Htt-NTF aggregation and neurotoxicity.