Soluble Membrane Protein Libraries: Design and use in Alzheimers drug discovery

描述（由申请人提供）：神经元突触是一种复杂的质膜，包含数千种蛋白质，其聚集功能定义了认知、感觉信息处理和运动控制。在阿尔茨海默病（AD）中，被称为淀粉样蛋白2衍生的扩散配体（ADDL）的毒素的突触靶向被认为诱导突触丧失和其他病理过程。Klein实验室和其他实验室的先前工作表明，ADDL靶向是特异性的，并且以组织，细胞和突触特异性的方式与受体介导的突触位点结合。然而，关于ADDL与神经元关联的机制的竞争假设也在文献中得到支持。目前，缺乏对神经ADDL结合的直接机制洞察是研究ADDL结合引发的途径和设计治疗策略以防止神经元ADDL靶向的进展的重要障碍。我们将采用新的，无细胞的策略来测试受体介导的ADDL结合的假设工程可溶性“膜蛋白库”分离的神经膜。这些库是通过将膜组分从异质生物膜转移到纳米盘中来创建的-纳米级，脂质双层的可溶性结构域被稳定蛋白质环包围。纳米盘允许一种至几种膜蛋白的受控掺入和稳定，并且它们作为用于研究ADDL结合的体外模型系统的使用代表了允许应用生物化学和生物物理工具来收集关于神经元ADDL关联的性质的直接的分子水平信息的使能技术。在目标1中，我们将确定ADDL和神经膜成分之间的分子相互作用范围，测试ADDL通过特定毒素受体攻击神经元的假设。为了实现这种分析，我们将开发和表征可溶性的基于纳米盘的膜蛋白文库，其概括神经元ADDL结合，如通过许多标志评估的，包括细胞群体之间的结合特异性和使用已知调节剂影响ADDL/纳米盘结合的能力。7在目标2中，作为体外ADDL结合测定的初始治疗应用，我们将开发和采用无细胞高通量测定用于潜在的AD治疗。所提出的测定评估小分子抑制ADDL与从生物膜分离的含有ADDL结合位点的纳米盘相互作用的能力。成功的化合物将在基于细胞的测定中验证ADDL结合和毒性。这里提出的纳米盘封装的膜蛋白库代表了一种新的解决方案，膜蛋白的不溶性和突触膜的复杂性的常年问题，允许在概述AD中ADDL诱导的突触病理学的机制基础的进展。 公共卫生相关性：目前许多药物通过与细胞表面的蛋白质结合而起作用。我们正在开发一种新方法，使研究这些蛋白质和发现新疗法变得更容易。在第一个应用中，我们将使用这种新方法来发现能够防止阿尔茨海默病相关毒素攻击大脑中细胞表面蛋白的化合物。

DESCRIPTION (provided by applicant): The neuronal synapse is a complex plasma membrane housing thousands of proteins whose aggregate functions define cognition, the processing of sensory information, and motor control. In Alzheimer's disease (AD), the synaptic targeting of toxins known as Amyloid 2-Derived Diffusible Ligands (ADDLs) is thought to induce synapse loss and other pathological processes. Previous work in the Klein lab and other labs suggests that ADDL targeting is specific and receptor-mediated - binding synaptic sites in a tissue-, cell-, and synapse-specific manner. However, competing hypotheses regarding the mechanism of ADDL association with neurons are also supported in the literature. Currently, the lack of direct mechanistic insight into neural ADDL binding is a significant barrier to progress in studying the pathways initiated by ADDL binding and designing therapeutic strategies to prevent neuronal ADDL targeting. We will employ novel, cell-free strategy to test the receptor-mediated hypothesis of ADDL binding by engineering soluble "membrane protein libraries" isolated from neural membranes. These libraries are created by transferring membrane components from heterogeneous biological membranes into nanodiscs - nanoscale, soluble domains of lipid bilayers encircled by a ring of stabilizing proteins. Nanodiscs allow the controlled incorporation and stabilization of one to several membrane proteins, and their use as an in vitro model system for studying ADDL binding represents an enabling technology allowing the application of biochemical and biophysical tools to gather direct, molecular-level information on the nature of neuronal ADDL association. 7 In Aim 1, we will determine the range of molecular interactions between ADDLs and constituents of neural membranes, testing the hypothesis that ADDLs attack neurons through specific toxin receptors. To enable this analysis, we will develop and characterize soluble, nanodisc-based, membrane protein libraries that recapitulate neuronal ADDL binding as evaluated by a number of hallmarks including binding specificity between cell populations and the ability to affect ADDL/nanodisc binding using known modulators. 7 In Aim 2, as an initial therapeutic application for an in vitro ADDL binding assay, we will develop and employ a cell-free high-throughput assay for potential AD therapeutics. The proposed assays evaluate the ability of small molecules to inhibit the interaction of ADDLs with nanodiscs containing ADDL binding sites isolated from biological membranes. Successful compounds will be validated in cell-based assays for ADDL binding and toxicity. The nanodisc-encapsulation of membrane protein libraries proposed here represents a novel solution to the perennial problems of membrane protein insolubility and synaptic membrane complexity, allowing progress in outlining the mechanistic basis of ADDL-induced synaptic pathology in AD. PUBLIC HEALTH RELEVANCE: Many current drugs work by binding to proteins at the surface of cells. We are developing a new method that will make it easier to study these proteins and discover new therapies. In a first application, we will use this new method to discover compounds capable of preventing Alzheimer's-related toxins from attacking cell-surface proteins in the brain.