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

描述（由申请人提供）：神经元突触是一个复杂的质膜，包含数千种蛋白质，其聚合功能定义认知，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衍生扩散配体（ADDLs）的毒素的突触靶向被认为会诱导突触丢失和其他病理过程。Klein实验室和其他实验室先前的工作表明，ADDL靶向是特异性的，并且以组织、细胞和突触特异性的方式靶向受体介导的结合突触位点。然而，关于ADDL与神经元关联机制的竞争性假设也在文献中得到支持。目前，缺乏对神经ADDL结合的直接机制认识是研究ADDL结合启动途径和设计治疗策略以防止神经元ADDL靶向的重大障碍。我们将采用新颖的无细胞策略，通过从神经膜中分离的工程可溶性“膜蛋白文库”来验证受体介导的ADDL结合假说。这些文库是通过将膜组分从异质生物膜转移到纳米圆盘中创建的-纳米级，脂质双分子层的可溶性结构域被一圈稳定蛋白包围。纳米盘可以控制一到几种膜蛋白的结合和稳定，它们作为研究ADDL结合的体外模型系统代表了一种使能技术，允许应用生化和生物物理工具来收集神经元ADDL结合本质的直接分子水平信息。在目标1中，我们将确定ADDLs与神经膜成分之间的分子相互作用范围，验证ADDLs通过特定毒素受体攻击神经元的假设。为了实现这一分析，我们将开发和表征可溶性的、基于纳米圆盘的膜蛋白文库，这些文库概括了神经元ADDL结合，并通过许多标志进行评估，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/纳米圆盘结合的能力。在目标2中，作为体外ADDL结合测定的初始治疗应用，我们将开发并采用无细胞高通量测定潜在的AD治疗方法。所提出的实验评估了小分子抑制ADDL与从生物膜分离的含有ADDL结合位点的纳米圆盘相互作用的能力。成功的化合物将在基于细胞的测定中验证ADDL的结合和毒性。本文提出的膜蛋白库的纳米圆盘封装为长期存在的膜蛋白不溶性和突触膜复杂性问题提供了一种新的解决方案，从而在概述adl诱导的AD突触病理的机制基础方面取得了进展。

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.