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: 10317950
• 负责人: Jeannie Chen
• 金额: $ 60.41万
• 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/10317950
• 关键词: Binding; Biochemical; Biological Assay; Biological Markers; Cells; Consensus; Cryoelectron Microscopy; Development; Disease; Epitopes; Exons; Foundations; Future; Goals; Huntington Disease; Huntington gene; Huntington protein; Lead; 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; Seeds; Structure; Testing; Therapeutic; Toxic effect; Work; aggregation pathway; conformer; design; inhibitor/antagonist; 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