Exploiting New Fibril Structures to Understand the Biophysical Basis for Oligomerization and Toxicity of Alpha-Synuclein

利用新的原纤维结构来了解 α-突触核蛋白寡聚化和毒性的生物物理基础

• 批准号: 10684133
• 负责人: Jonathan N Sachs
• 金额: $ 37.99万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684133
• 关键词: Affinity; Alzheimer' s Disease; Amino Acid Motifs; Amino Acid Sequence; Amino Acids; Back; Biological Assay; Biology; Biophysics; Catalogs; Cell Line; Cell model; Cells; Chemicals; Collaborations; Color; Communities; Computer Models; Coupled; Cytoprotection; Data; Dementia; Disease; Event; Experimental Designs; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Investigation; Kinetics; Label; Medicine; Methodology; Microscopy; Modeling; Molecular; Molecular Structure; Molecular Weight; Monitor; Morphologic artifacts; Mutation; Nature; Neurons; Neurosciences; Outcome; Paper; Parkinson Disease; Pathology; Pathway interactions; Positioning Attribute; Proteins; Protocols documentation; Publications; Recording of previous events; Research; Research Personnel; Resolution; Science; Series; Signal Transduction; Structure; System; Techniques; Technology; Testing; Therapeutic; Time; Total Internal Reflection Fluorescent; Toxic effect; Variant; Work; advanced simulation; algorithm development; alpha synuclein; beta pleated sheet; biophysical analysis; biophysical tools; cell type; cytotoxic; cytotoxicity; design; drug discovery; high throughput screening; inhibitor; innovation; insight; kinetic model; molecular modeling; molecular scale; monomer; mutant; neuron loss; new therapeutic target; novel; novel strategies; prevent; protein folding; protein misfolding; rational design; screening; small molecule; small molecule inhibitor; time use; tool

Abstract Research into the molecular basis of Parkinson’s Disease has recently undergone a dramatic shift to focus on toxic, early stage oligomers of α-Synuclein (aSyn). Understanding this promising new therapeutic target, a departure from research on insoluble fibrils, now requires biophysical insight about the misfolding of aSyn monomers and subsequent assembly of these toxic oligomers. These oligomer species are far less understood than fibrils, and more difficult to study, presenting a pressing challenge to biophysicists. The specific overall goal of the proposed work is to identify a subset of amino acid interactions within and between aSyn monomers that are most important in the assembly and toxicity of oligomers. Several new high- resolution structures of aSyn fibrils will be used as an exciting starting point to launch detailed investigations into the structural motifs that are present in the early stages of assembly. Based on strong preliminary results, we hypothesize that, despite their relative structural disorder, there exist robust, targetable structural motifs in early stage oligomers that persist through fibrilization. Additionally, a subset of those motifs is essential in determining toxicity: some promote toxic assemblies while others promote cytoprotective assemblies. High-resolution structures of early-stage oligomers will likely never be solved. Absent structures, our data will do the next best thing: it will point to specific motifs and residues that stabilize early-stage oligomers and that should be the focus of directed targeting campaigns. We have established a highly resolved technology (both temporally and spatially), time-resolved FRET, that allows us to study with great sensitivity the early-stages of aSyn aggregation in the cell. We will support these cellular observations with rigorous biophysical studies including 19F NMR, two-color TIRF microscopy and computational modeling. We will also utilize our established small molecule discovery technology in an innovative way to establish whether there are clear structural differences in oligomeric assemblies of the familial variants of aSyn, and whether these assemblies vary in differing neuronal cell lines. In sum, the proposal will provide the field with a significantly deeper understanding of the biophysical basis of aSyn oligomerization and will draw new correlations between key amino-acid residues, folding and toxicity.

摘要 帕金森病的分子基础研究最近经历了一个戏剧性的转变， α-突触核蛋白（aSyn）的毒性早期寡聚体。了解这个有前途的新治疗靶点， 从对不溶性纤维的研究出发，现在需要对aSyn的错误折叠进行生物物理学洞察 单体和这些有毒低聚物的后续组装。这些低聚物物种远不为人所知 比原纤维更难研究，这给生物药理学家带来了紧迫的挑战。 拟议工作的具体总体目标是确定氨基酸相互作用的子集， 在低聚物的组装和毒性中最重要的aSyn单体之间。几个新高- aSyn纤维的分辨率结构将被用作一个令人兴奋的起点，以展开详细的研究， 在组装的早期阶段出现的结构基序。根据初步的结果，我们 假设，尽管它们相对结构紊乱，但在早期存在稳健的、可靶向的结构基序， 通过原纤化持续存在的阶段低聚物。此外，这些图案的子集对于确定 毒性：一些促进毒性组装，而另一些促进细胞保护组装。高分辨率 早期低聚物的结构可能永远不会被解决。没有结构，我们的数据将做得最好 事情：它将指向稳定早期低聚物的特定基序和残基，这应该是重点 有针对性的目标定位活动。 我们已经建立了一个高分辨率的技术（时间和空间），时间分辨FRET， 使我们能够以极高的灵敏度研究细胞中aSyn聚集的早期阶段。我们将支持这些 通过严格的生物物理研究进行细胞观察，包括19 F NMR，双色TIRF显微镜和 计算建模我们还将利用我们建立的小分子发现技术， 方法来确定是否有明确的结构差异，在寡聚体组装的家族变异， aSyn，以及这些组件是否在不同的神经元细胞系中变化。 总之，该提案将使该领域对生物物理基础有更深入的了解 的aSyn寡聚化，并将绘制关键氨基酸残基，折叠和毒性之间的新的相关性。

项目成果

Advancements in a FRET Biosensor for Live-Cell Fluorescence-Lifetime High-Throughput Screening of Alpha-Synuclein.

α-突触核蛋白的活细胞荧光高通量筛选的FRET生物传感器中的进步。

• DOI: 10.1177/17590914231184086
• 发表时间: 2023-01
• 期刊: ASN NEURO
• 影响因子: 4.7
• 作者: Braun, Anthony R.;Kochen, Noah Nathan;Yuen, Samantha L.;Liao, Elly E.;Cornea, Razvan L.;Thomas, David D.;Sachs, Jonathan N.
• 通讯作者: Sachs, Jonathan N.