STRUCTURE-FUNCTION ANALYSIS OF SHIGA-LIKE TOXIN

志贺样毒素的结构功能分析

基本信息

批准号：
2517226
负责人：
CAROLYN J HOVDE
金额：
$ 11.13万
依托单位：
UNIVERSITY OF IDAHO
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-09-01 至 1999-08-31
项目状态：
已结题

项目摘要

The long range goals of this project are to understand the structure-function relationships of the Shiga toxin family, at the molecular level. All members of this class of toxins act by catalyzing the removal of a single adenine at position 4324 from ribosomal RNA. This N-glycosidic depurination inactivates the ribosome, inhibits protein synthesis, and causes target cell death. Holotoxins in this family consist of a single enzymatically active A subunit and multiple identical B subunits that bind the toxin to susceptible cells. The work described is focused on Shiga-like toxin type I (SLT-1) which our laboratory -uses as a representative model of this larger group of proteins. SLT is produced by the enterohemorrhagic Escherichia coli (EHEC) and has been strongly associated with human diarrhea syndromes, hemorrhagic colitis, and two extra-colonic complications, the hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Outlined here, are three approaches to characterize the active site of SLT-1 A subunit (SLT-IA) by (i) oligonucleotide-directed mutagenesis, which will be guided by sequence similarity among toxins in the ricin and Shiga family and computer modeling of the SLT-IA structure; (ii) random point mutagenesis of SLT-IA and selection for reduced toxicity in yeast, a eukaryotic host sensitive to wild-type toxin; and (iii) determining the 3-dimensional structure of enzymatically active SLT-IA and mutants of interest, which will require the purification and crystallization of recombinant SLT-IA and mutants. The fourth aim is to use SLT-specific monoclonal antibodies, synthetic peptides and site-directed mutation to identify and characterize the amino acid residues of SLT-IA and SLT-IB involved in holotoxin assembly. The fifth aim is to develop a murine model, using recombinant Citrobacter freundii which produce SLT, to determine the mechanism of pathogenesis in diseases associated with EHEC infection. Detailed structure-function information about SLT-1 is pertinent to bacterial pathogenesis, vaccine production, design of new therapies, and insights into endogenous regulatory enzymes yet to be characterized.

该项目的长期目标是了解志贺毒素家族的结构与功能关系分子水平。此类毒素的所有成员均通过催化作用发挥作用从核糖体 RNA 中去除位置 4324 处的单个腺嘌呤。这种 N-糖苷脱嘌呤使核糖体失活，抑制蛋白质合成，并导致靶细胞死亡。这个家族中的全毒素由单个酶活性 A 亚基和多个相同的亚基组成 B 亚基将毒素与易感细胞结合。所描述的工作专注于我们实验室使用的 I 型志贺毒素 (SLT-1) 作为这一较大蛋白质组的代表性模型。 SLT 是由肠出血性大肠杆菌（EHEC）产生，并已被与人类腹泻综合症、出血性结肠炎、以及两种结肠外并发症，即溶血性尿毒症综合征，以及血栓性血小板减少性紫癜。这里概述了三种方法通过 (i) 表征 SLT-1 A 亚基 (SLT-IA) 的活性位点寡核苷酸定向诱变，将由序列引导蓖麻毒素和志贺家族毒素与计算机之间的相似性 SLT-IA结构建模； (ii) SLT-IA的随机点诱变以及对酵母（一种对真核宿主敏感的酵母）毒性降低的选择野生型毒素； (iii) 确定 3 维结构酶活性 SLT-IA 和感兴趣的突变体，这需要重组SLT-IA及其突变体的纯化和结晶。第四个目标是使用SLT特异性单克隆抗体，合成肽和定点突变来识别和表征 SLT-IA 和 SLT-IB 的氨基酸残基参与全毒素组装。第五个目标是使用重组柠檬酸杆菌开发小鼠模型产生SLT的弗氏菌，以确定其发病机制与肠出血性大肠杆菌感染相关的疾病。详细结构功能有关 SLT-1 的信息与细菌发病机制、疫苗有关新疗法的生产、设计以及对内源性的见解调节酶尚待表征。