ROLE OF CHAIN LENGTH AND SEQUENCE CONTEXTS ON POLYGLUTAMINE OLIGOMERIZATION

描述(申请人提供)：亨廷顿病是一种毁灭性的神经退行性疾病，由亨廷顿蛋白(HTT)基因第一外显子CAG密码子扩展引起。其他八种蛋白质中类似的CAG重复扩增与八种不同的神经退行性疾病有关。在所有九种疾病中，CAG重复扩张编码蛋白质产物中的聚谷氨酰胺扩张，疾病的发病和严重程度与聚谷氨酰胺长度呈负相关，尽管这种相关性的数量性质对于九种疾病中的每一种都是不同的。多聚谷氨酰胺的膨胀最终形成不可溶的神经元包涵体，越来越多的证据表明，聚集机制和可溶低聚物物种直接与这九种疾病中的每一种选择性神经变性有关。多聚谷氨酰胺的膨胀破坏了它们的宿主蛋白的稳定性，并增加了蛋白质分解的可能性。蛋白质降解的片段由多聚谷氨酰胺束和侧翼N-末端和C-末端片段组成。聚谷氨酰胺伸展侧翼的N-末端和C-末端片段对每种疾病相关蛋白都是独特的。均聚谷氨酰胺的聚集驱动力随着链长的增加而变得更强，而自然产生的N-末端和C-末端侧翼序列调节了这一驱动力。我们的目标是了解疾病相关蛋白中聚谷氨酰胺扩展两侧的序列如何调节聚谷氨酰胺固有的、依赖于长度的构象偏好和聚集机制。我们的方法是基于新颖的原子模拟和一组体外实验的组合。我们最近的结果与这样的假设是一致的，即自然发生的侧翼序列可以作为“守门人”来抑制聚集倾向区域的内在聚集倾向。因此，目前的提议是以以下假设为指导的：疾病相关蛋白中自然存在的侧翼序列可以充当守门人，以减少多谷氨酰胺束的内在聚集倾向。这种影响可以通过导致聚谷氨酰胺长度增加的扩张突变来克服。此外，把关机制可能因侧翼顺序而异，从而导致把关效率的差异。我们将使用新的原子模拟和体外实验相结合的方法来表征1)不同自然发生的末端侧翼序列内的构象变化以及这些变化之间的耦合和分子内界面内易于聚集的聚谷氨酰胺区域作为聚谷氨酰胺长度的函数的隔离/暴露程度；2)自然发生的侧翼序列是否真正是守门人，并量化这些序列作为聚谷氨酰胺长度的函数调节聚集的程度。精确了解侧翼序列和聚谷氨酰胺扩张之间的偶联机制将使我们能够确定抑制聚集介导的毒性和神经退行性变途径的靶点。

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.