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

• 批准号: 10441571
• 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
• 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/10441571
• 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 of activation protein; Membrane Proteins; Modeling; Modification; Molecular; Molecular Structure; N-terminal; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathologic; Pathology; Persons; Pharmacology; Physiological; Play; Polysaccharides; Post-Translational Protein Processing; Process; Proteins; Reporting; Research; Resolution; Role; Seeds; Single Nucleotide Polymorphism; Specificity; Structure; Surface; Synapses; System; Testing; Therapeutic; Variant; Work; alpha synuclein; design; extracellular; glycosylation; insight; live cell imaging; monomer; mutant; novel; protein aggregation; receptor; small molecule; small molecule therapeutics; success; synaptic function; synaptogenesis; synergism; therapeutically effective; transmission process; uptake

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.

项目总结/摘要 α-突触核蛋白是一种小的可溶性神经元蛋白，是路易体聚集体的主要成分 是帕金森病的标志虽然机械细节还没有很好地理解， 新出现的证据表明，α-突触核蛋白毒性形式的细胞间传递是疾病的基础 传播我们的实验室最近已经确定了复杂的N-连接聚糖作为细胞内化的介质， α-突触核蛋白的单体和聚集体形式都带有N-末端乙酰基， 蛋白质的修饰。我们特别鉴定了神经元糖蛋白neurexin 1β， 以聚糖依赖性方式驱动α-突触核蛋白的内化。我们研究的目标是 表征了α-Synuclein与neurexin 1β结合的结构基础，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 α-突触核蛋白结合neurexin 1β后的细胞内化。我们的假设是细胞间 αS的传递依赖于与neurexin 1β的相互作用，这些相互作用的选择性是 依赖于N-末端乙酰基赋予的α-突触核蛋白的瞬时结构变化。到 为了研究这一假设，我们制定了三个具体目标，包括： 与neurexin 1β结合的α-突触核蛋白的结构特征，包括定义最小α-突触核蛋白构建体 结合所需的（目的1）;确定与neurexin 1β结合导致细胞凋亡的机制。 α-突触核蛋白的内化（目的2）;并了解α-突触核蛋白与neurexin结合的功能影响 1β（目标3）。为了实现这些目标，我们将在体外进行粗晶粒和高分辨率的结构， 表征α-突触核蛋白：neurexin 1β复合物，并使用活细胞成像来量化 α-突触核蛋白和内化的α-突触核蛋白引发内源性α-突触核蛋白聚集的能力。我们 将对比WT单体、PD相关点突变体和α-突触核蛋白的纤维状形式。通过这个 我们希望通过这项研究来表征参与α-突触核蛋白病理学传播的关键相互作用， 帕金森病，以及深入了解α-突触核蛋白的结构特征：neurexin 1β 配合物最后，通过我们的研究，对α-突触核蛋白：neurexin 1β相互作用进行了表征。 这些研究可能为帕金森病的治疗提供新的靶点，并作为识别新的 小分子疗法