Structural basis and physiological consequences of alpha-Synuclein binding to neurexin 1beta

α-突触核蛋白与神经毒素 1β 结合的结构基础和生理学后果

基本信息

批准号：
10677814
负责人：
Elizabeth Rhoades
金额：
$ 57.45万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10677814
关键词：
Acetylation Affinity Automobile Driving Binding Brain Cell membrane Cells Characteristics Coculture Techniques Communities Complex Data Deposition Disease Fluorescence Resonance Energy Transfer Future Genes Global Change Glycoproteins Goals Grain In Vitro Inherited Kinetics Lewy Bodies Link Mass Spectrum Analysis Measures Mediating Mediator Membrane Proteins Modeling Modification Molecular Molecular Structure N-terminal Neurodegenerative Disorders Neurons Parkinson Disease Pathologic Pathology Persons Physiological Play Polysaccharides Post-Translational Protein Processing Process Proteins Reporting Research Resolution Role Single Nucleotide Polymorphism Specificity Structure Surface Synapses System Testing Therapeutic Variant Work alpha synuclein design extracellular glycosylation in vivo insight live cell imaging monomer mutant novel pharmacologic presynaptic protein aggregation receptor small molecule small molecule therapeutics success synaptic function synaptogenesis synergism therapeutically effective transmission process uptake

项目摘要

Project Summary/Abstract α-Synuclein is a small, soluble neuronal protein that is the primary component of the Lewy body aggregates that are the hallmark of Parkinson's disease. While the mechanistic details are not yet well-understood, emerging evidence suggests that cell-to-cell transmission of toxic forms of α-Synuclein is the basis of disease propagation. Our lab has recently identified complex N-linked glycans as mediators of cellular internalization of both monomer and aggregate forms of α-Synuclein bearing an N-terminal acetyl group, a physiological modification of the protein. We specifically identified the neuronal glycoprotein neurexin 1β as capable of driving internalization of α-Synuclein in a glycan-dependent manner. The goal of our proposed research is to characterize the structural basis of α-Synuclein binding to neurexin 1β, the role of both N-terminal acetylation and glycosylation in conferring specificity in this interaction, and determine the molecular mechanisms resulting cellular internalization of α-Synuclein following binding neurexin 1β. Our hypothesis is that cell-to-cell transmission of αS is dependent on interactions with neurexin 1β and that the selectivity in these interactions is dependent on transient structural changes in α-Synuclein conferred by the N-terminal acetyl group. To investigate this hypothesis, we have developed three specific aims with the following goals: determine the structural features of α-Synuclein bound to neurexin 1β, including defining a minimal α-Synuclein construct required for binding (Aim 1); determine the mechanisms by which binding to neurexin 1β results in cellular internalization of α-Synuclein (Aim 2); and understand the functional impact of α-Synuclein binding to neurexin 1β (Aim 3). To achieve these goals, we will carry out in vitro coarse grain and high resolution structural characterization of α-Synuclein:neurexin 1β complexes and use live-cell imaging to quantify internalization of α-Synuclein and the ability of internalized α-Synuclein to seed aggregation of endogenous α-Synuclein. We will contrast WT monomer, PD-associated point mutants and fibrillar forms of α-Synuclein. Through this research we expect to characterize key interactions involved in propagation of α-Synuclein pathology in Parkinson's disease, as well as to gain insight into the structural features of α-Synuclein:neurexin 1β complexes. Ultimately, the characterization of α-Synuclein: neurexin 1β interactions carried out through our studies may provide a new target for Parkinson's disease treatment and serve as the basis identifying novel small molecule therapeutics.

项目概要/摘要 α-突触核蛋白是一种小型可溶性神经元蛋白，是路易体聚集体的主要成分这是帕金森病的标志，虽然其机制细节尚不清楚，新的证据表明，有毒形式的 α-突触核蛋白在细胞间的传播是疾病的基础我们的实验室最近发现了复杂的 N 连接聚糖作为细胞内化的介质。带有 N 末端乙酰基的 α-突触核蛋白的单体和聚集体形式，是一种生理学我们特别鉴定了神经元糖蛋白神经素 1β 能够以聚糖依赖性方式驱动 α-突触核蛋白的内化我们提出的研究的目标是表征 α-突触核蛋白与神经毒素 1β 结合的结构基础，以及 N 末端乙酰化的作用和糖基化赋予这种相互作用的特异性，并确定产生的分子机制结合神经素 1β 后 α-突触核蛋白的细胞内化。我们的假设是细胞间的相互作用。 αS 的传输取决于与神经素 1β 的相互作用，并且这些相互作用的选择性为依赖于 N 末端乙酰基所赋予的 α-突触核蛋白的瞬时结构变化。为了研究这一假设，我们制定了三个具体目标，其目标如下：与神经毒素 1β 结合的 α-突触核蛋白的结构特征，包括定义最小的 α-突触核蛋白构建体结合所需（目标 1）；确定与神经蛋白 1β 结合导致细胞 α-突触核蛋白的内化（目标 2）；并了解 α-突触核蛋白与神经毒素结合的功能影响 1β（目标3）为了实现这些目标，我们将进行体外粗晶和高分辨率结构。 α-突触核蛋白：neurexin 1β 复合物的表征并使用活细胞成像来量化 α-突触核蛋白和内化 α-突触核蛋白促进内源 α-突触核蛋白聚集的能力。将通过此对比 WT 单体、PD 相关点突变体和纤维形式的 α-突触核蛋白。我们希望通过研究来表征 α-突触核蛋白病理学传播中涉及的关键相互作用帕金森病，以及深入了解 α-突触核蛋白：neurexin 1β 的结构特征最终，α-突触核蛋白：神经毒素 1β 相互作用的表征是通过我们的进行的。研究可能为帕金森病的治疗提供新的靶点，并作为识别新型帕金森病的基础小分子疗法。