Identifying the Interactions between Animal Toxins and Human nAChRs: The Role of Snake PLA2 in Interacting with nAChR alpha Subunits

项目概要/摘要 烟碱乙酰胆碱受体 (nAChR) 参与多种基本生理过程， 这些受体的功能障碍与许多人类疾病有关，例如阿尔茨海默病和 帕金森病。人类 nAChR 的结构和功能已得到广泛研究；然而， 不同 nAChR 亚基的组合形成各种 nAChR 亚型及其胞外区域 结合结构多样的配体，对高通量（HT）和系统性分析提出了挑战 nAChR 的研究。幸运的是，动物毒素提供了一种独特的工具来研究毒素的结构和功能。 nAChR，因为它们经过数百万年的自然选择微调，呈现出高 对特定蛋白质（例如 nAChR）的特异性和选择性。我们的长期目标是确定通用的 或与人类 nAChR 相互作用的选择性动物毒素，并阐明其机制基础 他们的互动的基础。为了实现这个目标，本应用的总体目标是建立HT 识别蛇毒素和 nAChR 亚基之间相互作用的方法以及破译 毒素在与人类 nAChR 相互作用中的作用，提出以下三个具体目标：1) 生成蛇毒素 ORFeome 库。独特的蛇毒素转录本开放阅读框（ORF）将 从医学上重要的蛇 (Crotalus atrox) 的现有毒腺 cDNA 文库中鉴定出来，并且 单独连接到“供体”载体中以创建可直接用于 与任何人类蛋白质（包括 nAChR）的相互作用检测。 2）确定蛇的相互作用 具有人类 nAChR 亚基的毒素。蛇毒素与人类 nAChR 亚基之间的相互作用 重点检测毒液磷脂酶 A2 (PLA2) 和 nAChR α 亚基，将通过以下方式鉴定 利用 HT 酵母双杂交 (HT-Y2H) 系统，然后进行共亲和纯化/质谱分析 （美联社/女士）。 3) 破译毒液毒素在与 nAChR 亚基相互作用中的作用。我们将开发一个HT 确定 PLA2 等蛇毒素是否对人类具有激动剂或拮抗剂作用的药理学测定 nAChR。 nAChR，例如α1β1γδ、α3β4、α4β2和α7，将被单独转染到人体细胞中 例如 HEK293，用于测量 PLA2-nAChR 引起的钙动力学和膜电位 互动。研究结果预计将产生影响，因为：1）它将提供宝贵的资源 供科学界、大学教职员工和学生进行后续生物学研究和生物医学 应用程序； 2）它将为HT和结构和系统研究提供独特的工具 nAChR 的功能。此外，该项目将为本科生创造大量机会 提高他们在生物医学科学研究技能。

PROJECT SUMMARY/ABSTRACT Nicotinic acetylcholine receptors (nAChRs) are involved in a variety of fundamental physiological processes, and dysfunction in these receptors is associated with many human disorders such as Alzheimer’s and Parkinson’s diseases. The structure and function of human nAChRs are extensively studied; however, the combinations of different nAChR subunits making various nAChR subtypes and their extracellular regions binding a structurally diverse range of ligands, pose challenges in high-throughput (HT) and systematic investigation of nAChRs. Fortunately, animal toxins provide a unique tool to study the structure and function of nAChR because they have been fine-tuned through millions of years of natural selection to present high specificity and selectivity towards specific proteins such as nAChRs. Our long-term goal is to identify universal or selective animal toxins that interact with human nAChRs, and elucidate the mechanistic underpinnings underlying their interactions. To achieve this goal, the overall objective of this application is to establish HT approaches for identifying the interactions between snake toxins and nAChRs subunits, as well as deciphering the role of toxins in interacting with human nAChRs by proposing the following three specific aims: 1) Generate a snake toxin ORFeome library. The unique snake toxin transcript open-reading frames (ORFs) will be identified from the existing venom gland cDNA library of a medically important snake (Crotalus atrox), and individually ligated into a “donor” vector to create a snake toxin ORFeome library that can be directly used for the interaction detections with any human proteins including nAChRs. 2) Determine the interactions of snake toxins with human nAChR subunits. The interactions between snake toxins and human nAChR subunits, with focus on the detection between venom phospholipase A2 (PLA2) and nAChR α subunits, will be identified by utilizing a HT yeast two-hybrid (HT-Y2H) system, followed by co-affinity purification/mass spectrometry (AP/MS). 3) Decipher the role of venom toxins in interacting with nAChR subunits. We will develop a HT pharmacological assay to determine if snake toxins such as PLA2 act as agonist or antagonist against human nAChRs. The nAChRs such as α1β1γδ, α3β4, α4β2, and α7, will be individually transfected into human cells such as HEK293 for measuring the calcium dynamics and membrane potential elicited by PLA2-nAChR interactions. The research results are expected to be impactful because: 1) it will provide valuable resources for the scientific community, university faculty and students for subsequent biological research and biomedical applications; and 2) it will provide a unique tool for HT and systematic investigation of the structure and function of nAChRs. In addition, the project will create numerous opportunities for undergraduate students to improve their research skills in biomedical science.