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首页> 外文学位 >Structure/activity relationship studies and target identification methodologies for the o-ACTX-1 neurotoxins.
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Structure/activity relationship studies and target identification methodologies for the o-ACTX-1 neurotoxins.

机译：o-ACTX-1神经毒素的结构/活性关系研究和目标识别方法。

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摘要

The o-atracotoxin-1 (o-ACTX-1) neurotoxins are 36--37-residue peptides found in the venom of all species of Australian funnel-web spiders examined to date. The prototypic family member, o-ACTX-Hv1a, is lethal to a wide range of insects and potently antagonizes high-voltage activated (HVA) calcium channels---a component of the insect nervous system never before targeted by a commercially available insecticide. Yet various lines of evidence suggest that o-ACTX-Hv1a is nontoxic to vertebrate organisms and has little or no detectable activity against many, if not all, vertebrate nervous system components. Thus, continued characterization of the o-ACTX-1's should enable better understanding of the insect nervous system and perhaps also facilitate development of selective agents which kill insect pests with minimal collateral damage to the environment.; Described herein is the development of a plasmid-based, bacterial expression system for o-ACTX-Hv1a. Also described are procedures which yielded detailed characterizations of the structure and bioactive surface of o-ACTX-Hv1a. These included heteronuclear NMR experiments which enabled the toxin structure to be determined with extremely high precision, and scanning mutagenesis which facilitated identification of several functionally critical regions in the toxin structure.; Also described is the development of methods and strategies which may enable definitive identification of the molecular target of the o-ACTX-1's: First, stocks of Drosophila melanogaster carrying inducible transgenes encoding o-ACTX-Hv1a were genetically engineered and propagated. The behavior of these stocks in response to transgene activity was consistent with antagonism of the Dmca1D subfamily of calcium channels, offering one of several lines of evidence presented and/or discussed herein that these channels represent the primary targets of the o-ACTX-1 toxins. Second, facile methods were developed for labeling o-ACTX-Hv1a with tracers and/or affinity tags without compromising its biological activity. Third, Anopheles gambiae cDNAs encoding subunits of a Dmca1D family calcium channel were subcloned. A heterologous calcium channel expression system based on these cDNAs could enable definitive confirmation that the o-ACTX-1 family selectively targets Dmca1D channels and could provide a valuable resource both for toxin-target interaction studies and for discovery of novel small molecule insecticides.
机译：迄今为止，o-atracotoxin-1（o-ACTX-1）神经毒素是在澳大利亚漏斗蜘蛛所有物种的毒液中发现的36--37残基肽。原型家族成员o-ACTX-Hv1a对多种昆虫具有致死性，并有效拮抗高压激活（HVA）钙通道-昆虫神经系统的组成部分，之前从未被市售杀虫剂靶向。然而，各种证据表明，o-ACTX-Hv1a对脊椎动物没有毒性，并且对许多（如果不是全部）脊椎动物神经系统成分几乎没有或没有可检测的活性。因此，对o-ACTX-1的持续表征应该能够更好地了解昆虫的神经系统，也许还可以促进选择性杀虫剂的开发，这些杀灭害虫的杀虫剂对环境的损害最小。本文描述了用于o-ACTX-Hv1a的基于质粒的细菌表达系统的开发。还描述了产生o-ACTX-Hv1a的结构和生物活性表面详细特征的方法。这些包括异核NMR实验，该实验使毒素结构能够以极高的精度确定，扫描诱变则有助于鉴定毒素结构中的几个功能关键区域。还描述了方法和策略的发展，这些方法和策略可以使o-ACTX-1的分子靶标得以最终鉴定：首先，对携带果蝇的果蝇的果蝇进行遗传工程改造和繁殖，这些果蝇将携带编码o-ACTX-Hv1a的可诱导转基因。这些原种对转基因活性的响应行为与钙通道Dmca1D亚家族的拮抗作用一致，为本文提供和/或讨论的这些证据代表了o-ACTX-1毒素的主要靶点提供了若干证据之一。其次，开发了简便的方法来用示踪剂和/或亲和标签标记o-ACTX-Hv1a，而不会损害其生物学活性。第三，亚克隆编码Dmca1D家族钙通道亚单位的冈比亚按蚊cDNA。基于这些cDNA的异源钙通道表达系统可以明确证实o-ACTX-1家族选择性靶向Dmca1D通道，并且可以为毒素-靶相互作用研究和发现新型小分子杀虫剂提供宝贵的资源。

著录项

  • 作者

    Tedford, Hugo W., Jr.;

  • 作者单位

    University of Connecticut.;

  • 授予单位 University of Connecticut.;
  • 学科 Biology Molecular.; Biology Neuroscience.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 227 p.
  • 总页数 227
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;神经科学;
  • 关键词

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