STRUCTURE FUNCTION STUDY OF MT3 TOXIN

MT3毒素的结构功能研究

• 批准号: 7073018
• 负责人: KRISHNA BAKSI
• 金额: $ 18.52万
• 依托单位: UNIVERSIDAD CENTRAL DEL CARIBE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7073018
• 关键词: CHO cells; aminoacid; analog; biotechnology; chimeric proteins; intermolecular interaction; muscarinic receptor; protein engineering; protein structure; protein structure function; receptor binding; reptile poison; toxin; transfection /expression vector; yeasts

DESCRIPTION (provided by applicant): Muscarinic acetylcholine receptors (mAChRs) are coupled to G-proteins and activate different second messengers and ion channels. Currently, there are five subtypes of mAChRs (MrM5), and are differentially distributed throughout the body. mAChRs are involved in a variety of disorders such as Schizophrenia, Parkinson's disease (PD), Alzheimer's disease (AD), cardiac arrhythmia, glaucoma, irritable bowel syndrome and asthma. Irrespective of the technological developments, the study of functions and characterization of mAChR subtypes has been lagging behind due to lack of highly selective agonists and antagonists. Several toxins which recognize the mAChRs have been purified and sequenced from the venom of mamba snakes of the genus Dendroaspis. The sequences of the toxins are homologous, but their mode of action and selectivity for mAChR subtypes are different. For example, MT3 toxin shows higher affinity for M4 receptors, and lower affinity for M! receptors, whereas, MT7 is specific for Iv^ receptors. The structure-function relationship of MT3 toxin is essential to establish the critical amino acid residues for the binding to the M4 receptors. Synthetic gene for MT3 toxin and the expression system are available in my laboratory for such studies. The hypothesis of this proposal is that the functional site of MT3 toxin requires multiple amino acid residues which are present in all the three loops of the toxin for its subtype specificity as well as for its action as an antagonist. I, therefore, propose to study the structure-function relationship of MT3 toxin, and the specific aims of my proposal are: SPECIFIC AIM 1: Production of MT3/MT7 chimeras and MT3 analogues: This will be achieved by the following experiments: (i) Synthesis MT3 toxin gene, and expression of MT3 toxin in Pichia pastoris using pPICZaA expression vector, and will be purified by the standard methods; (ii) Production of MT3/MT7 chimeras and MT3 analogue; and (iii) The structure-function of MT3/MT7 chimeras and MT3 analogues. The chimeras/analogues will be screened for their binding to Mi-M5 receptors expressed individually in CHO cells using [3H]-NMS binding assay. SPECIFIC AIM 2: The mode of interactions of MT3 toxin analogues with its receptors. MT3 analogues with higher selectivity for M4 receptors will be subjected for the agonist/antagonist/allosteric interactions using CHO cells expressing M4 receptors. The completion of this study will establish the specific amino acid residues responsible for MT3 toxin's subtype specificity. The long-term goal of this proposal is to design, and to develop therapeutic agents based on the three-dimensional template on which smaller molecule mimics can be designed for different disorders involving mAChRs.

描述（由申请人提供）：毒蕈碱乙酰胆碱受体（mAChR）与G蛋白偶联并激活不同的第二信使和离子通道。目前，mAChR (MrM5) 有 5 种亚型，并且在全身分布有差异。 mAChR 与多种疾病有关，例如精神分裂症、帕金森病 (PD)、阿尔茨海默病 (AD)、心律失常、青光眼、肠易激综合征和哮喘。无论技术如何发展，由于缺乏高选择性的激动剂和拮抗剂，对 mAChR 亚型的功能和表征的研究一直滞后。几种识别 mAChR 的毒素已从树蛇属曼巴蛇的毒液中纯化并测序。毒素的序列是同源的，但它们的作用方式和对 mAChR 亚型的选择性不同。例如，MT3毒素对M4受体的亲和力较高，而对M受体的亲和力较低！受体，而MT7对Ivk受体具有特异性。 MT3 毒素的结构-功能关系对于建立与 M4 受体结合的关键氨基酸残基至关重要。我的实验室有MT3毒素的合成基因和表达系统可供此类研究。该提议的假设是，MT3 毒素的功能位点需要多个氨基酸残基，这些氨基酸残基存在于毒素的所有三个环中，以实现其亚型特异性及其作为拮抗剂的作用。因此，我提议研究MT3毒素的结构与功能关系，我的提议的具体目标是： 具体目标1：MT3/MT7嵌合体和MT3类似物的生产：这将通过以下实验来实现：（i）合成MT3毒素基因，并使用pPICZaA表达载体在毕赤酵母中表达MT3毒素，并通过以下方法进行纯化： 标准方法； (ii) MT3/MT7嵌合体和MT3类似物的生产； (iii) MT3/MT7嵌合体和MT3类似物的结构-功能。将使用[ 3 H]-NMS结合测定筛选嵌合体/类似物与CHO细胞中单独表达的Mi-M5受体的结合。具体目标 2：MT3 毒素类似物与其受体的相互作用模式。对 M4 受体具有更高选择性的 MT3 类似物将使用表达 M4 受体的 CHO 细胞进行激动剂/拮抗剂/变构相互作用。这项研究的完成将确定负责 MT3 毒素亚型特异性的特定氨基酸残基。该提案的长期目标是设计和开发基于三维模板的治疗剂，在此基础上可以针对涉及 mAChR 的不同疾病设计更小的分子模拟物。