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

识别动物毒素与人类 nAChR 之间的相互作用：蛇 PLA2 在与 nAChR α 亚基相互作用中的作用

• 批准号: 10818654
• 负责人: YING JIA
• 金额: $ 4.18万
• 依托单位: UNIVERSITY OF TEXAS RIO GRANDE VALLEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10818654
• 关键词: Affinity Chromatography; Agonist; Alzheimer' s Disease; Animals; Asp snake; Binding; Biological Assay; Calcium; Cells; Communities; Consultations; Crotalus; Detection; Disease; Faculty; Functional disorder; Generations; Gland; Goals; Human; Human Resources; Individual; Investigation; Ion Channel Gating; Libraries; Ligands; Ligation; Literature; Mass Spectrum Analysis; Measures; Medical; Membrane Potentials; Molecular; Natural Selections; Neuroglia; Neurons; Neurosciences; Nicotinic Receptors; Open Reading Frames; Outcome; Parkinson Disease; Phospholipase A2; Physiological Processes; Protein Isoforms; Proteins; Reader; Research; Research Personnel; Resources; Role; Science; Snake Venoms; Snakes; Specificity; Structure; Students; Testing; Texas; Toxin; Training; Transcript; Transfection; Universities; Venoms; Yeasts; alpha-bungarotoxin receptor; antagonist; biological research; cDNA Library; detection assay; extracellular; improved; member; molecular dynamics; pharmacologic; receptor; skills; tool; undergraduate student; vector; yeast two hybrid system

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.

项目摘要/摘要 烟碱乙酰胆碱受体（NACHR）参与了多种基本生理过程， 这些受体的功能障碍与许多人类疾病有关，例如阿尔茨海默氏症和 帕金森的疾病。人NACHR的结构和功能是广泛研究的。但是， 不同NACHR亚基的组合，使各种NACHR亚型及其细胞外区域的组合 结合结构上的潜水配体范围，高通量（HT）和系统的挑战 NACHRS的投资。幸运的是，动物毒素提供了研究的独特工具来研究 NACHR是因为它们已经通过数百万年的自然选择进行了微调，以表现出高度 对特定蛋白质（例如NACHRS）的特异性和选择性。我们的长期目标是确定通用 或与人NACHR相互作用并阐明机械基础的选择性动物毒素 基础他们的互动。为了实现这一目标，本应用的总体目标是建立HT 识别蛇毒素与NACHRS亚基之间相互作用的方法 通过提出以下三个特定目的，毒素在与人NACHR相互作用中的作用：1） 生成蛇毒素Orfeome库。独特的蛇毒素成绩单开放阅读框（ORF）将 可以从医学上重要的蛇（crotalus atrox）的现有毒腺cDNA文库中识别出来， 单独连接到“供体”向量，以创建可直接用于的蛇毒素orfeome库 与包括NACHR的任何人类蛋白质的相互作用检测。 2）确定蛇的相互作用 带有人NACHR亚基的毒素。蛇毒素与人类NACHR亚基之间的相互作用与 专注于毒液磷脂酶A2（PLA2）和NACHRα亚基之间的检测，将通过 使用HT酵母双杂交（HT-Y2H）系统，然后进行共亲密纯化/质谱法 （AP/MS）。 3）破译毒素在与NACHR亚基相互作用中的作用。我们将发展一个HT 药理学评估以确定蛇毒素（如Pla2）是对人类的激动剂还是对人类的拮抗剂的作用 nachrs。 NACHR（例如α1β1γδ，α3β4，α4β2和α7）将分别转化为人类细胞 例如用于测量PLA2-NACHR引起的钙动力学和膜电位的HEK293 互动。预计研究结果将具有影响力，因为：1）它将提供宝贵的资源 对于科学界，大学的教职员工和学生的生物学研究和生物医学 申请； 2）它将为结构的HT和系统投资提供独特的工具 NACHR的功能。此外，该项目将为本科生创造许多机会 提高他们在生物医学科学方面的研究技能。