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.

该项目的远距离目标是了解 Shiga毒素家族的结构功能关系，在分子水平。这类毒素的所有成员通过催化作用从核糖体RNA的位置4324处去除单个腺嘌呤。这种N-糖苷脱硫使核糖体灭活，抑制蛋白质合成，并导致靶细胞死亡。这个家庭中的全毒素由单个酶活性的亚基和多个相同的 B将毒素与易感细胞结合的亚基。描述的工作专注于我们实验室的类似Shiga的毒素I型（SLT-1）作为这种较大的蛋白质的代表性模型。 SLT是由Enterohemorrhagic Escherichia Coli（EHEC）生产的与人腹泻综合征，出血性结肠炎密切相关，以及两个外侧色并发症，溶血性尿毒症综合征，以及血栓性血小板减少紫菜。这里概述了三种方法通过（i）表征SLT-1亚基（SLT-ia）的活动位点寡核苷酸指导的诱变将以序列为指导 Ricin和Shiga家族和计算机中的毒素之间的相似性 SLT-IA结构的建模；（ii）SLT-IA的随机点诱变和选择降低酵母的毒性，真核宿主对到野生型毒素；（iii）确定的3维结构具有酶活性的SLT-IA和感兴趣的突变体，这将需要重组SLT-IA和突变体的纯化和结晶。第四个目的是使用SLT特异性单克隆抗体，合成肽和位置突变，以识别和表征 SLT-IA和SLT-IB的氨基酸残基参与蛋白毒素组装。第五个目标是使用重组柠檬酸杆菌开发鼠模型产生SLT的Freundii，以确定发病机理与EHEC感染相关的疾病。详细的结构功能有关SLT-1的信息与细菌发病机理有关生产，新疗法的设计以及对内生的见解调节酶尚待表征。