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.

抽象的 帕金森氏病分子基础的研究最近发生了巨大的转变 关于α-突触核蛋白（ASYN）的有毒早期低聚物。了解这个有前途的新治疗靶 偏离对不溶性原纤维的研究，现在需要关于Asyn错误折叠的生物物理见解 这些有毒的低聚物的单体和随后的组装。这些低聚物物种知之甚少 比原纤维更难研究，对生物物理学家提出了紧迫的挑战。 拟议工作的具体总体目标是确定内部氨基酸相互作用的子集 在寡聚体组装和毒性中最重要的Asyn单体之间。几个新的高级 Asyn Fibrils的分辨率结构将被用作令人兴奋的起点，以启动详细研究 在组装早期阶段存在的结构基序。基于强大的初步结果，我们 假设，在它们的相对结构障碍中，早期存在强大的，可定位的结构基序 通过纤维化持续存在的阶段低聚物。此外，这些基序的子集对于确定 毒性：有些促进有毒组件，而另一些则促进细胞保护组件。高分辨率 早期低聚物的结构可能永远不会解决。缺乏结构，我们的数据将取得下一个最好的 某事：它将指出特定的图案并保留稳定早期低聚物的特定主题，这应该是重点 定向目标运动。 我们已经建立了一项高度解决的技术（无论是临时还是空间上），时间分辨的货物，该技术是 使我们能够以极大的敏感性研究细胞中ASYN聚集的早期阶段。我们将支持这些 细胞观察进行了严格的生物物理研究，包括19F NMR，两色TIRF显微镜和 计算建模。我们还将利用我们已建立的小分子发现技术来创新 确定家庭变体的寡聚组件是否存在明显的结构差异 Asyn以及这些组件是否在区分神经元细胞系中有所不同。 总而言之，该提案将为该领域提供对生物物理基础的更深入的了解 Asyn寡聚化，并将在关键氨基酸的保留，折叠和毒性之间提出新的相关性。

项目成果

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

• 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.