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

描述(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，这些蛋白质的聚合功能定义了认知、感觉信息的处理和运动控制。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(ADDLS)的毒素的突触靶向被认为导致突触丢失和其他病理过程。Klein实验室和其他实验室之前的工作表明，Add1靶向是组织、细胞和突触特异的、受体介导的突触结合位点。然而，文献中也支持关于ADD1与神经元联系机制的相互竞争的假说。目前，缺乏对神经ADD1结合的直接机制的了解是研究ADDL结合启动的通路和设计防止神经元ADD1靶向的治疗策略的重要障碍。我们将使用新的、无细胞的策略，通过设计从神经膜中分离出的可溶“膜蛋白文库”来验证受体介导的ADD1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盘上创建的-纳米级的可溶性域的脂质双层被一环稳定的蛋白质包围。纳米盘允许一到几个膜蛋白的受控掺入和稳定，它们作为研究ADD1结合的体外模型系统的使用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关于神经元ADD1结合性质的直接的、分子水平的信息。7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通过特定的毒素受体攻击神经元的假设。为了进行这一分析，我们将开发和表征可溶的、基于纳米盘的膜蛋白文库，这些文库概括了神经元ADT1结合的情况，通过一系列特征进行评估，包括细胞群体之间的结合特异性和使用已知调节剂影响ADD1/纳米盘结合的能力。7在目标2中，作为体外Add1结合试验的初步治疗应用，我们将开发并使用一种无细胞高通量试验来潜在的AD治疗。所提出的检测方法评估了小分子抑制ADDLS与含有从生物膜中分离出的ADTL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ADDL结合和毒性测试中得到验证。本文提出的膜蛋白纳米盘包埋库为解决膜蛋白不溶性和突触膜复杂性这一长期存在的问题提供了一种新的解决方案，为阐明ADD1诱导AD突触病理的机制提供了新的途径。

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.