Unravelling the Cause of Parkinson's Disease in Molecular Detail

从分子细节上揭示帕金森病的病因

• 批准号: 2439628
• 金额: --
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2439628
• 关键词: Unravelling; Cause; Parkinson; Disease; Molecular

A-Synuclein is a 140 amino acid, 14 kDa, intrinsicallydisordered protein. Neuronal aggregates of a-Synuclein arethe hallmarks of Parkinson's Disease. The protein containsa series of imperfect KTKEGV motifs carrying a net positivecharge spread across the first 25 N-terminal residues andcentral NAC region, covering residues 25-100 whilst the Cterminal is negatively charged. NMR chemical shiftassignments of the monomer in solution have beenpublished. A-Synuclein associates with lipid membranesurfaces in a sequential manner, with an N-terminal anchorstrongly bound and the NAC membrane sensing region lessstrongly bound. On binding, both these regions adopt anamphipathic helical conformation. Exposure of themembrane bound NAC region has been implicated inaberrant misfolding, oligomerisation, aggregation,membrane insertion and pore formation. A-Synucleinoligomers are highly toxic to cells and act as seedingspecies in intercellular spread. Solid state and Cryo-EMstudies have revealed the structure of the end stage fibrilsbut mechanistic studies of the membrane bound monomerto oligomer transition have proven challenging although theNMR solution structure of a-Synuclein bound to an SLSmicelle has been published.In this proposal we will exploit the small size of detergentmicelles to facilitate acquisition of high resolution solutionstate NMR data. In previous studies a-Synuclein has beenuniformly labelled with NMR active nuclei. Here we willemploy a selective labelling approach using Cysteine pointmutations to install NMR active labels at strategic pointsdistributed along the linear sequence of the protein. Thiswill allow us to visualise in molecular detail the assemblyof a-Synuclein molecules in a way not possible withuniformly labelled material due to severe spectroscopicoverlap when multiple copies of protein are present. Wewill confirm the integrity of the modified Synucleins withstandard biophysical analysis. We will generate a panel ofsingle and doubly labelled proteins suitable for inter andintra-molecular nOe experiments revealing the mechanismof the earliest events in dimer, trimer, tetramer and highorder oligomer formation and their morphology viz:parallel, antiparallel, in or out of register.Further we will develop customised NMR probes of differentvector length and orientation providing data on both shortand long range interactions in the lipid bound state.Finally we will apply our approach to naturally occurringfamilial mutant a-Synucleins known to have differentiatedmembrane binding affinity which is correlated with diseasepathology in terms of onset of age and rate of progression.

A-突触核蛋白是一种由140个氨基酸组成、分子量为14 kDa的蛋白质。α-突触核蛋白的神经元聚集是帕金森病的标志。该蛋白含有一系列不完全的KTKEGV基序，携带一个净正电荷，分布在N端的前25个残基和中央NAC区，覆盖残基25-100，而C端带负电荷。已发表了该单体在溶液中的NMR化学位移谱。A-突触核蛋白以顺序方式与脂质膜表面结合，N-末端锚定强烈结合，NAC膜感应区结合不那么强烈。在结合时，这两个区域都采用两亲性螺旋构象。膜结合NAC区的暴露与异常错误折叠、寡聚化、聚集、膜插入和孔形成有关。A-突触核蛋白寡聚体对细胞具有高度毒性，并在细胞间扩散中充当种子物种。固态和Cryo-EM研究已经揭示了纤维末端的结构，但是膜结合单体到低聚物转变的机理研究已经证明是具有挑战性的，尽管已经发表了结合到SLS胶束的α-Synuclein的NMR溶液结构，在这个提议中，我们将利用小尺寸的洗涤剂胶束来促进高分辨率溶液态NMR数据的获取.在以前的研究中，α-突触核蛋白已经被NMR活性核均匀标记。在这里，我们将采用一种选择性标记的方法，使用半胱氨酸点突变安装NMR活性标签的战略点分布沿着线性序列的蛋白质。这将使我们能够在分子细节上可视化α-突触核蛋白分子的组装，而当存在多个蛋白质拷贝时，由于严重的光谱重叠，这种方式不可能使用均匀标记的材料。我们将用标准的生物物理学分析来证实修饰过的突触核蛋白的完整性。我们将产生一组适合分子间和分子内nOe实验的单标记和双标记蛋白质，揭示二聚体、三聚体、四聚体和高级低聚体形成中最早事件的机制及其形态，即：平行，反平行，我们将进一步开发不同载体长度和方向的定制NMR探针，提供脂质结合状态下短程和长程相互作用的数据。最后，我们将把我们的方法应用于自然发生的家族性突变a-已知突触核蛋白具有不同的膜结合亲和力，这与发病年龄和进展速度方面的疾病病理学相关。