Molecular Mechanism of Huntingtin Misfolding and its Inhibition by Designed and Cellular Proteins

摘要 亨廷顿病（HD）是许多神经变性疾病之一， 聚集蛋白是一种致病机制。HD是由亨廷顿蛋白中的多聚谷氨酰胺扩增引起的。 蛋白质，这使得它和它的天然存在的外显子1片段（Httex 1），更容易聚集。我们有 表明Httex 1聚集是一个逐步的过程，其中单体引起不同的聚集 在形成原纤维之前的中间体。虽然有很好的共识，Httex 1聚合起着关键作用， 在疾病发病机制中的作用，对Httex 1聚集中间体的3D结构知之甚少， 每种构象异构体对毒性的贡献该领域的一个主要障碍是难以获得 这些构象异构体的生物化学表征的同质群体。我们最近发现， 稳定制备，并表征了Httex 1聚集过程中不同的中间体。我们建议 通过确定关键构象（α-螺旋低聚物和非捆绑原纤维）的结构， 研究可以抑制错误折叠和毒性的不同机制。使用我们的一系列不同的 构象，我们也希望获得详细的洞察分子伴侣如何识别HTTEX 1构象。通过 结合EPR，固态和溶液NMR，cryo-EM和细胞毒性测定，我们的团队处于独特的位置 成功实现这些目标。在目标1中，我们将结合联合收割机EPR，NMR，cryo-EM和计算 改进，以确定来自具有不同Q长度的Httex 1蛋白的未捆绑原纤维的结构。通过 了解这些有毒构象异构体的结构，我们可以在未来的努力，旨在寻找生物标志物， 聚集抑制剂。最早的错误折叠中间体，α-螺旋低聚物的结构，将是 在目标2A中确定。这将使用EPR、溶液NMR和cryo-EM进行。我们还得到了一种纤维粘合剂 来自小的、多聚化的N17 Q7肽，其有效地抑制Httex 1聚集。具体目标2B测试 假设该结合剂通过干扰初级和/或次级接种来抑制聚集。此外，委员会认为， 我们将优化抑制剂并测试其在细胞环境中保护免受毒性的能力。具体目标3 决定了分子伴侣如何识别Httex 1错误折叠。使用生物化学方法，EPR， NMR和cryo-EM，我们将确定分子伴侣（DNAJB 1和DNAJB 6）结合的分子机制 通过确定它们结合的Httex 1构象和它们识别的表位来识别Httex 1。

Abstract Huntington disease (HD) is one of many neurodegenerative diseases wherein accumulation of misfolded, aggregated protein is a pathogenic mechanism. HD is caused by polyglutamine expansions in the huntingtin protein which make it and its naturally occurring exon 1 fragment (Httex1), more aggregation prone. We have shown that Httex1 aggregation is a stepwise process, wherein the monomer gives rise to different aggregation intermediates prior to formation of fibrils. Although there is good consensus that Httex1 aggregation plays a key role in disease pathogenesis, less is known about the 3D structures of Httex1 aggregation intermediates and how each conformer contributes to toxicity. A major obstacle in the field has been the difficulty in obtaining homogeneous population of these conformers for their biochemical characterization. We have recently identified, stably prepared, and characterized different different intermediates during Httex1 aggregation. We propose to extend this work by determining the structure of key conformers (α-helical oligomer and unbundled fibril) and by investigating different mechanism by which misfolding and toxicity can be inhibited. Using our array of different conformers, we also expect to obtain detailed insight into the how chaperones recognize Httex1 conformers. By combining EPR, solid-state and solution NMR, cryo-EM, and cell toxicity assays, our team is in a unique position to successfully accomplish these goals. In Aim 1, we will combine EPR, NMR, cryo-EM and computational refinement to determine the structure of unbundled fibrils from Httex1 proteins with different Q-lengths. By learning about the structures of these toxic conformers, we enable future efforts aimed at finding biomarkers and aggregation inhibitors. The structure of the earliest misfolding intermediate, the α-helical oligomer, will be determined in Aim 2A. This will be done using EPR, solution NMR, and cryo-EM. We also obtained a fibril binder from small, multimerized N17Q7 peptides which potently inhibits Httex1 aggregation. Specific aim 2B tests the hypothesis that this binder inhibits aggregation by interfering with primary and/or secondary seeding. Moreover, we will optimize the inhibitor and test its ability to protect from toxicity in a cellular setting. Specific aim 3 determines how chaperones recognize Httex1 misfolding. Using a combination of biochemical methods, EPR, NMR and cryo-EM, we will identify the molecular mechanism by which chaperones (DNAJB1 and DNAJB6) bind to Httex1 by determining which Httex1 conformers the they bind to and which epitope they are recognizing.