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

亨廷顿蛋白错误折叠的分子机制及其设计和细胞蛋白的抑制

• 批准号: 10686966
• 负责人: Jeannie Chen
• 金额: $ 57.24万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-17 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686966
• 关键词: Binding; Biochemical; Biological Assay; Biological Markers; Cells; Consensus; Cryoelectron Microscopy; Development; Disease; Epitopes; Exons; Foundations; Future; Goals; Huntington Disease; Huntington gene; Learning; Length; Methods; Molecular; Molecular Chaperones; N-terminal; Neurodegenerative Disorders; Oranges; Pathogenesis; Pathogenicity; Peptides; Play; Population; Positioning Attribute; Process; Proline-Rich Domain; Proteins; Research; Role; Structure; Testing; Therapeutic; Toxic effect; Work; aggregation pathway; alpha helix; conformer; design; inhibitor; insight; interest; misfolded protein; monomer; polyglutamine; prevent; protein aggregation; solid state; three dimensional structure

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.

摘要 亨廷顿病(HD)是许多神经退行性疾病之一，其中错误折叠堆积， 聚集蛋白是一种致病机制。HD是由狩猎过程中多聚谷氨酰胺的膨胀引起的 使其及其自然产生的外显子1片段(Httex 1)更容易聚集的蛋白质。我们有 表明Httex 1的聚集是一个逐步的过程，其中单体引起不同的聚集 纤维形成之前的中间产物。尽管有一个很好的共识，即Httex 1聚合起到了关键作用 在疾病发病机制中的作用，对Httex 1聚集中间体和 每一种构象是如何造成毒性的。这一领域的一个主要障碍是难以获得 这些异构体的同质种群，用于其生化特性。我们最近发现， 稳定地制备，并在Httex 1聚集过程中表征不同的中间体。我们建议 通过确定关键构象(α-螺旋低聚物和非结合纤维)的结构和通过 研究抑制错误折叠和毒性的不同机制。使用我们的不同数组 除了构象外，我们还希望对伴侣如何识别Httex 1构象有详细的了解。通过 结合EPR、固体和溶液核磁共振、低温EM和细胞毒性分析，我们的团队处于独特的地位 为了成功地实现这些目标。在目标1中，我们将结合EPR、核磁共振、低温电磁和计算 精炼以确定不同Q-长度的Httex-1蛋白的非结合纤维的结构。通过 了解这些有毒构象的结构，我们使未来的努力旨在寻找生物标记物和 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、溶液核磁共振和冷冻-EM来完成。我们还得到了一种纤维粘合剂 来自小的、多聚体的N17Q7肽，它有效地抑制Httex 1的聚集。特定目标2B测试 假设这种粘结剂通过干扰一次和/或二次播种来抑制聚集。此外， 我们将优化该抑制剂，并在细胞环境中测试其保护毒性的能力。具体目标3 确定伴侣如何识别Httex 1错误折叠。使用生化方法的组合，EPR， 核磁共振和冷冻-EM，我们将确定分子伴侣(DNAJB1和DNAJB6)结合的机制 通过确定它们与哪些Httex 1构象结合以及它们识别哪个表位来识别Httex 1。

项目成果

What makes functional amyloids work?

• DOI: 10.1080/10409238.2022.2113030
• 发表时间: 2022-08
• 期刊: Critical reviews in biochemistry and molecular biology
• 影响因子: 6.5

Solid-state NMR of paired helical filaments formed by the core tau fragment tau(297-391).

• DOI: 10.3389/fnins.2022.988074
• 发表时间: 2022
• 期刊: FRONTIERS IN NEUROSCIENCE
• 影响因子: 4.3
• 作者: Al-Hilaly, Youssra K. K.;Hurt, Connor;Rickard, Janet E. E.;Harrington, Charles R. R.;Storey, John M. D.;Wischik, Claude M. M.;Serpell, Louise C. C.;Siemer, Ansgar B. B.
• 通讯作者: Siemer, Ansgar B. B.