Blocking Cellular Intoxication by Bacterial Toxins

阻止细菌毒素引起的细胞中毒

• 批准号: 6653079
• 负责人: Steven R. Blanke
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6653079
• 关键词: Clostridium botulinum; acidity /alkalinity; biotechnology; bioterrorism /chemical warfare; botulinum toxins; disulfide bond; inhibitor /antagonist; membrane permeability; mutant; protein engineering; protein protein interaction; recombinant proteins

DESCRIPTION (provided by applicant): BoNTs are a dangerous bioterrorism threat due to their extreme potency and lethality, as well as their ease of production and transport. If untreated, poisoning by the BoNTs can progress to flaccid paralysis and death due to respiratory failure. However, timely post-exposure intervention can limit the effects of the circulating toxin. The overall, long-term research objective is to generate a novel class of therapeutics that can be administered to individuals who have been poisoned by BoNT. The strategy, as described in this application, will be to develop dominant-negative mutants of one form of the toxin, BoNT/A, which will interact with and inactivate complexes of wild type toxin. BoNT/A is composed of two defined fragments. The H chain facilitates the transport of a second toxin fragment, the L chain, into target cells. The strategy is based on the hypothesis that transport of the BoNT/A L chain, which is an essential step in the cellular intoxication mechanism, is mediated by pH-triggered unfolding and membrane insertion of toxin oligomeric complexes. Experimental and computational approaches will be used to develop a model for the mechanism of H chain-mediated membrane transport of the L chain. This model will be tested by altering the amino acid sequence of the H chain in a manner predicted to interfere with membrane transport. Specifically, intramolecular disulfide linkages will be engineered within the H chain to limit movement of the polypeptide backbone as a result of pH triggered conformational changes. Moreover, positively charged amino acids will be introduced throughout the H chain, which would be predicted to be unfavorable for membrane insertion of the BoNT/A H chain. Wild type and mutant forms of the H chain will be expressed as recombinant proteins, and each mutant will be tested for dominant-negative inhibitory activity in the presence of wild type toxin. Simultaneously, structure-function relationships important for translocation will be identified as an important prerequisite for fu1ure design of dominant-negative based inhibitors. A milestone of this work will be the identification of one or more dominant-negative BoNT/A mutants that will block action of wild type toxin using in vitro model systems. The results from this research will establish the groundwork and justification for future development and in vivo testing of these novel inhibitors using established animal models.

描述（由申请人提供）：由于其极端的效能和致死性以及其易于生产和运输，因此BONT是一种危险的生物恐怖威胁。如果未经治疗，骨会中毒会因呼吸衰竭而导致脆性瘫痪和死亡。但是，及时的暴露后干预可以限制循环毒素的影响。总体而言，长期的研究目标是生成一种新型的治疗剂，这些治疗剂可以用于被BONT中毒的个体进行。如本应用所述，该策略将是开发一种形式的毒素，BONT/A形式的显性阴性突变体，该突变体将与野生型毒素的复合物相互作用并灭活复合物。 BONT/A由两个定义的片段组成。 H链促进了第二个毒素片段（L链）进入靶细胞。该策略基于以下假设：BONT/A L链的运输是细胞中毒机制中必不可少的步骤，是由pH触发的展开和膜插入毒素寡聚体复合物介导的。实验和计算方法将用于开发用于L链H链介导的膜转运机理的模型。该模型将通过预测干扰膜转运的方式来改变H链的氨基酸序列。具体而言，分子内二硫键将在H链中进行设计，以限制pH触发构象变化的结果。此外，将在整个H链中引入带正电荷的氨基酸，预计这对于膜插入BONT/A H链会不利。 H链的野生型和突变形式将表示为重组蛋白，并且在存在野生型毒素的情况下，将测试每个突变体的显性阴性抑制活性。同时，对易位重要的结构 - 功能关系将被确定为基于主导阴性抑制剂的FU1URE设计的重要先决条件。这项工作的一个里程碑将是鉴定一个或多个主要的阴性BONT/A突变体，该突变体将使用体外模型系统阻止野生型毒素的作用。这项研究的结果将为未来发展和体内测试这些新型抑制剂使用既定的动物模型建立基础和理由。