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

项目摘要/摘要 α-突触核蛋白是一种小的、可溶的神经元蛋白，是路易小体聚集体的主要成分 这些都是帕金森氏症的特征。虽然机制细节还没有被很好地理解， 新出现的证据表明，有毒形式的α-突触核蛋白的细胞间传播是疾病的基础 传播。我们的实验室最近发现复杂的N-连接多聚糖是细胞内化的介体 带有N-末端乙酰基的α-突触核蛋白的单体和聚集体形式，生理上 蛋白质的修饰。我们特别鉴定了神经糖蛋白Neurexin 1β能够 以糖依赖的方式驱动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Neurein 1β结合的结构基础，N-末端乙酰化的作用 和糖基化在这种相互作用中赋予特异性，并确定导致的分子机制 α-突触核蛋白在结合β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，这些相互作用中的选择性是 依赖于由N-末端乙酰基引起的α-突触核蛋白的瞬时结构变化。至 研究这一假设，我们制定了三个具体目标，目标如下：确定 α-突触核蛋白与β结合的结构特征，包括定义一个最小的α-突触核蛋白结构 结合所需(目标1)；确定与Neurexin 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Neurein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和高分辨率的结构 α-突触核蛋白：Neurexin 1β复合体的表征及活细胞成像定量研究 α-突触核蛋白和内化α-突触核蛋白对内源性α-突触核蛋白种子聚集的能力。我们 将对比WT单体、PD相关的点突变和纤维形式的α-突触核蛋白。通过这件事 我们期望描述α-突触核蛋白病理传播中涉及的关键相互作用的研究 帕金森氏病，以及深入了解α-突触核蛋白：Neurexin 1β的结构特征 复合体。最终，α-突触核蛋白：Neurexin 1β相互作用的特征通过我们的 研究可能为帕金森氏病的治疗提供新的靶点，并为确定新的 小分子疗法。

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.