Exploiting new fibril structures to understand the biophysical basis for oligomerization and toxicity of alpha-Synuclein

摘要 对帕金森氏病分子基础的研究最近经历了戏剧性的转变 关于α-突触核蛋白(ASyn)的毒性早期寡聚体。了解这一有希望的新治疗靶点， 脱离了对不溶性纤维的研究，现在需要对aSyn错误折叠的生物物理洞察力 单体和这些有毒低聚物的后续组装。对这些低聚物种类的了解要少得多。 比纤维更难研究，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和内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原纤的分辨结构将作为一个令人兴奋的起点来展开详细的研究 在组装的早期阶段出现的结构主题。基于强劲的初步结果，我们 假设，尽管它们的结构相对混乱，但在早期存在健壮的、有针对性的结构主题 在纤维化过程中持续存在的阶段齐聚物。此外，这些主题的子集在确定 毒性：一些促进有毒集合体，而另一些促进细胞保护集合体。高分辨率 早期低聚物的结构很可能永远不会被解决。如果没有结构，我们的数据将表现得仅次于 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 有针对性的目标活动。 我们已经建立了一种高度分辨的技术(在时间和空间上)，时间分辨的FRET， 使我们能够非常敏感地研究细胞内aSyn聚集的早期阶段。我们将支持这些 用严格的生物物理研究进行细胞观察，包括19F核磁共振，双色TIRF显微镜和 计算建模。我们还将利用我们成熟的小分子发现技术在创新的 确定家族性变种的寡聚体组装是否存在明显结构差异的方法 ASyn，以及这些组件在不同的神经细胞系中是否有所不同。 总之，该提案将使该领域对生物物理基础有更深入的了解。 并将得出关键氨基酸残基、折叠和毒性之间的新关联。

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.