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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

抽象的对帕金森病分子基础的研究最近发生了焦点的巨大转变关于 α-突触核蛋白 (aSyn) 的有毒早期寡聚物的研究，了解这一有前途的新治疗靶点，a 偏离了对不溶性原纤维的研究，现在需要对 aSyn 错误折叠的生物物理学见解人们对这些有毒低聚物的单体和随后的组装知之甚少。比原纤维更难研究，这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和内部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单体之间的一些新的高- aSyn 原纤维的解析结构将作为一个令人兴奋的起点来展开详细的研究基于强有力的初步结果，我们确定了组装早期阶段存在的结构图案。研究发现，尽管它们的结构相对无序，但早期存在强大的、可靶向的结构基序。此外，这些基序的一个子集在确定过程中至关重要。毒性：一些促进有毒组装，而另一些则促进高分辨率组装。早期低聚物的结构可能永远无法解决，如果没有结构，我们的数据将是次优的。事情：它将指向稳定早期寡聚体的特定基序和残基，这应该是焦点定向目标活动。我们已经建立了一种高分辨率技术（时间和空间），即时间分辨 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 19F NMR、双色 TIRF 显微镜和我们还将利用我们已建立的小分子发现技术进行创新。确定家族变体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 aSyn 寡聚化的研究，并将得出关键氨基酸残基、折叠和毒性之间的新相关性。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

用于活细胞荧光寿命高通量筛选 α-突触核蛋白的 FRET 生物传感器的进展。

DOI：
发表时间：
2023
期刊：
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