STRUCTURE FUNCTION STUDY OF MT3 TOXIN

描述(申请人提供)：M受体(MAChRs)与G蛋白偶联，激活不同的第二信使和离子通道。目前，mAChRs有五种亚型(MrM5)，分布各异。MAChRs与多种疾病有关，如精神分裂症、帕金森氏病(PD)、阿尔茨海默病(AD)、心律失常、青光眼、肠易激综合征和哮喘。无论技术发展如何，由于缺乏高度选择性的激动剂和拮抗剂，对mAChR亚型的功能和特性的研究一直处于滞后状态。几种识别mAChRs的毒素已经从Dendroaspis属的曼巴蛇的毒液中纯化并测序。这些毒素的序列是同源的，但它们对mAChR亚型的作用方式和选择性不同。例如，MT3毒素对M4受体具有较高的亲和力，而对M！受体，而MT7对IV受体是特异的。MT3毒素的结构-功能关系是确定与M4受体结合的关键氨基酸残基的关键。MT3毒素的合成基因和表达系统在我的实验室可以用于这样的研究。这一假设是，MT3毒素的功能部位需要多种氨基酸残基，这些氨基酸残基存在于毒素的所有三个环中，因为其亚型特异性以及作为拮抗剂的作用。因此，我建议研究MT3毒素的结构和功能关系，我的建议的具体目的是：特异性目的1：制备MT3/MT7嵌合体和MT3类似物：这将通过以下实验实现：(I)合成MT3毒素基因，并在毕赤酵母中利用pPICZaA表达载体表达MT3毒素，并用标准方法进行纯化；(Ii)制备MT3/MT7嵌合体和MT3类似物；以及(Iii)MT3/MT7嵌合体和MT3类似物的结构与功能。用[~3H]-NMS结合试验筛选嵌合体/类似物与CHO细胞中单独表达的Mi-M5受体的结合。特异性目的2：MT3毒素类似物与其受体的相互作用方式。对M4受体具有较高选择性的MT3类似物将通过表达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导致MT3毒素亚型特异性的特定氨基酸残基。这项提议的长期目标是设计和开发基于三维模板的治疗剂，在该模板上可以为涉及mAChRs的不同疾病设计更小的分子模拟物。

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.