ROLE OF CHAIN LENGTH AND SEQUENCE CONTEXTS ON POLYGLUTAMINE OLIGOMERIZATION

描述（由申请人提供）：亨廷顿氏病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 CAG密码子扩增引起。其他8种蛋白质中类似的CAG重复扩增与8种不同的神经退行性疾病有关。在所有9种疾病中，CAG重复扩增编码蛋白产物中的聚谷氨酰胺扩增，疾病的发病和严重程度与聚谷氨酰胺长度呈负相关，尽管这种相关性的定量性质对每种疾病都不同。聚谷氨酰胺扩增最终以不溶性神经元包涵体结束，越来越多的证据表明，聚集机制和可溶性寡聚物物种与九种疾病中的每种疾病的选择性神经变性直接相关。聚谷氨酰胺扩增会破坏宿主蛋白的稳定性，增加蛋白水解的可能性。蛋白质水解的片段由聚谷氨酰胺束和两侧的N端和c端片段组成。聚谷氨酰胺拉伸两侧的N端和c端片段对每种疾病相关蛋白都是独特的。均聚聚谷氨酰胺的聚集驱动力随着链长的增加而增强，自然存在的N端和c端侧链序列调节了这种驱动力。我们的目标是了解疾病相关蛋白中聚谷氨酰胺扩增的序列如何调节聚谷氨酰胺固有的、长度依赖的构象偏好和聚集机制。我们的方法是基于新的原子模拟和一组体外实验的结合。我们最近的研究结果与自然发生的侧翼序列可以作为“看门人”抑制聚集易发区域的内在聚集倾向的假设是一致的。因此，目前的建议以以下假设为指导：疾病相关蛋白中自然存在的侧翼序列可以作为看门人，减少聚谷氨酰胺束的内在聚集倾向。这种效应可以通过扩展突变来克服，从而增加聚谷氨酰胺的长度。此外，守门机制可能因侧翼序列而异，从而导致守门效率的差异。我们将使用新的原子模拟和体外实验的组合来表征1)不同自然发生的末端侧翼序列的构象变化，以及这些变化与分子内界面中易于聚集的聚谷氨酰胺区域的隔离/暴露程度之间的耦合，作为聚谷氨酰胺长度的函数；2)如果自然发生的侧翼序列是真正的守门人，并量化其程度这些序列作为聚谷氨酰胺长度的函数调节聚集。对侧翼序列和聚谷氨酰胺扩增之间耦合机制的精确理解将使我们能够确定抑制聚集介导的毒性和神经变性途径的靶标。

DESCRIPTION (provided by applicant): Huntington's disease is a devastating neurodegenerative disease caused by CAG codon expansion in exon 1 of the huntingtin (htt) gene. Similar CAG repeat expansions in eight other proteins are associated with eight different neurodegenerative diseases. In all nine diseases, the CAG repeat expansions encode polyglutamine expansions in the protein products, and the onset and severity of disease are inversely correlated with the polyglutamine length although the quantitative nature of this correlation is different for each of the nine disorders. Polyglutamine expansions end up in insoluble neuronal inclusions and there is growing evidence that the mechanisms of aggregation and the soluble oligomeric species are directly linked to selective neurodegeneration in each of the nine diseases. Polyglutamine expansions destabilize their host proteins and increase the likelihood of proteolysis. Fragments of proteolysis consist of polyglutamine tracts and flanking N- and C-terminal segments. The N- and C-terminal segments that flank the polyglutamine stretch are unique to each disease-related protein. Driving forces for aggregation of homopolymeric polyglutamine becomes stronger with increasing chain length and naturally occurring N- and C-terminal flanking sequences modulate this driving force. Our goal is to understand how sequences that flank polyglutamine expansions in disease-related proteins modulate the intrinsic, length-dependent conformational preferences and aggregation mechanisms of polyglutamine. Our approaches are based on a combination of novel atomistic simulations and a panel of in vitro experiments. Our recent results are consistent with the hypothesis that naturally occurring flanking sequences can act as "gatekeepers" to suppress intrinsic aggregation propensities of aggregation-prone regions. Therefore, the current proposal is guided by the following hypothesis: Naturally occurring flanking sequences in disease-related proteins can act as gatekeepers to decrease the intrinsic aggregation tendencies of polyglutamine tracts. This effect can be overcome by expansion mutations that lead to increased polyglutamine lengths. Additionally, gatekeeping mechanisms likely vary with flanking sequence, giving rise to differences in gatekeeping efficiencies. We will use a combination of novel atomistic simulations and in vitro experiments to characterize 1) conformational changes within different naturally occurring terminal flanking sequences and the coupling between these changes and the degree of sequestration / exposure of aggregation-prone polyglutamine regions within intramolecular interfaces as a function of polyglutamine length and 2) if naturally occurring flanking sequences are bona fide gatekeepers and to quantify the degree to which these sequences modulate aggregation as a function of polyglutamine length. Precise understanding of the mechanisms of coupling between flanking sequences and polyglutamine expansions will allow us to identify targets for inhibition of routes to aggregation-mediated toxicity and neurodegeneration. PUBLIC HEALTH RELEVANCE: There is clear connection between aggregation and the onset of devastating polyglutamine expansion diseases such as Huntington's disease. The role of flanking sequences in modulating aggregation is of direct relevance to the progression of polyglutamine expansion diseases. Mechanistic studies proposed here have a direct bearing on the development of drugs that inhibit the gain of function associated with polyglutamine aggregation.