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

项目摘要/摘要 烟碱型乙酰胆碱受体(NAChRs)参与多种基本的生理过程， 这些受体的功能障碍与许多人类疾病有关，如阿尔茨海默氏症和 帕金森氏症。人们对nAChRs的结构和功能进行了广泛的研究；然而， 不同nAChR亚基的组合形成不同的nAChR亚型及其胞外区 结合一系列结构不同的配体，在高通量(HT)和系统 NAChRs的研究。幸运的是，动物毒素为研究细胞的结构和功能提供了独特的工具。 NAChR，因为它们经过数百万年的自然选择微调，呈现出高度的 对特定蛋白质的特异性和选择性，如nAChRs。我们的长期目标是确定普遍的 或与人类nAChRs相互作用的选择性动物毒素，并阐明其机制基础 它们相互作用的基础。为了实现这一目标，本应用程序的总体目标是建立HT 蛇毒素与nAChRs亚基相互作用的鉴定方法及破译 毒素在与人的nAChRs相互作用中的作用提出了以下三个具体目标：1) 产生一种蛇毒素或Feome文库。独特的蛇毒转录开放阅读框(ORF)将 从一种具有重要药用价值的蛇(Crotalus Trex)的现有毒腺cDNA库中鉴定出，以及 单独连接到“供体”载体，以创建蛇毒素或Feome文库，可直接用于 这种相互作用可以检测到包括nAChRs在内的任何人类蛋白质。2)确定蛇的相互作用 含有人类nAChR亚基的毒素。蛇毒素与人nAChR亚基的相互作用 重点检测蛇毒磷脂酶A2(PLA2)和nAChRα亚基之间的关系，将通过 利用高温酵母双杂交(HT-Y2H)系统，然后进行共亲和纯化/质谱分析 (AP/MS)。3)破译毒素与nAChR亚基相互作用的作用。我们将开发一种超音速技术 确定PLA2等蛇毒素对人类是激动剂还是拮抗剂的药理试验 NAChRs。α-1-β-1-γδ，α-3-β-4、α-4-β-2和α-7等nAChR将被单独导入人细胞 如HEK293用于测量PLA2-nAChR引起的钙动力学和膜电位 互动。预计研究成果将产生影响，因为：1)它将提供宝贵的资源 为科学界、大学教职员工和学生提供后续的生物研究和生物医学 2)它将为超临界流体力学和系统的研究提供一个独特的工具。 NAChRs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方面的研究技能。

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.