Genetic Analysis of Cholera Toxin Structure and Function

描述（改编自申请人摘要）：约15亿 腹泻每年造成400万5岁以下儿童死亡 500 - 700万例霍乱病例造成约10万人死亡。霍乱毒素 (CT)霍乱弧菌引起的大量水样腹泻。 产肠毒素大肠大肠杆菌（ETEC）导致高达20%的肠道疾病， 发展中国家，并产生称为LTI和LTII的不耐热肠毒素 在结构和功能上与CT密切相关。当前最佳的 针对霍乱的疫苗只能在短期内提供适度的保护， 在美国没有许可证，也没有疫苗 人类使用ETEC。CT和相关肠毒素是有效的免疫原， 粘膜佐剂，它们也被广泛用作研究 异源三聚体G蛋白在信号转导中的作用， 神经节苷脂的内吞作用和囊泡运输，映射和/或 神经通路的消融以及许多其他细胞功能。我们研究 CT结构和功能，并利用LTI和LTII进行比较研究， 探索它们之间功能差异的分子基础。我们漫长 术语目标是阐明CT生物活性的分子基础 和相关的肠毒素，并利用这些知识来设计新的 预防或治疗肠毒性的基于结构的疫苗和治疗剂 - 是的CT、LTI或LTII也作为疫苗成分被广泛研究， 佐剂或免疫调节剂来预防或治疗与 肠毒性大肠杆菌。CT结构和功能的重要问题 还不了解的包括识别和表征： 激活CT-A1的催化能力的构象变化， CT全毒素的切口和减少; CT-A1上决定其 与作为ADP核糖基化底物的Gsalpha/beta/gamma相互作用， 用ADP-核糖基化因子（ARF）作为催化活性的刺激物; CT-A和CT-B的特征使它们能够自发地组装成CT 全毒素;肠毒素与质膜结合的机制 受体决定它们在靶细胞内的运输;以及通过 CT-A1从ER转移到细胞质， 细胞内靶向并引起毒性。在下一个项目期间， 使用各种各样的新方法，从微生物学，遗传学，生物化学， 细胞生物学和结构生物学来研究这些重要的电流 关于霍乱毒素的结构和功能的问题。

DESCRIPTION (Adapted from the Applicant's Abstract): Approximately 1.5 billion cases of diarrhea cause 4 million deaths annually in children under 5 years old, and 5-7 million cases of cholera cause about100,000 deaths. Cholera toxin (CT) from Vibrio cholerae causes the massive watery diarrhea of cholera. Enterotoxigenic E. coli (ETEC) cause up to 20 percent of diarrheal disease in developing countries, and produce heat-labile enterotoxins called LTI and LTII that are closely related to CT in structure and function. The best current vaccines against cholera provide only moderate protection for short periods of time and are not licensed in the United States, and there are no vaccines for human use against ETEC. CT and related enterotoxins are potent immunogens and mucosal adjuvants, and they are also used widely as tools to investigate the role of heterotrimeric G proteins in signal transduction, the role of gangliosides in endocytosis and vesicular trafficking, the mapping and/or ablation of neural pathways, and many other cell functions. We study the structure and function of CT and use LTI and LTII in comparative studies to explore the molecular basis for functional differences between them. Our long term goals are to elucidate the molecular basis for biological activities of CT and related enterotoxins, and to use that knowledge to design novel structure-based vaccines and therapeutics to prevent or treat enterotoxic diarrheas. CT, LTI or LTII are also being studied widely as vaccine components, adjuvants or immunomodulators to prevent or treat diseases unrelated to enterotoxic diarrheas. Important issues concerning structure and function of CT that are not yet understood include identifying and characterizing: conformational changes that activate the catalytic capacity of CT-A1 after nicking and reduction of CT holotoxin; motifs on CT-A1 that determine its interactions with Gsalpha/beta/gamma as a substrate for ADP ribosylation and with ADP-ribosylation factors (ARFs) as stimulators of catalytic activity; features of CT-A and CT-B that enable them to assemble spontaneously into CT holotoxin; mechanisms by which binding of enterotoxins to plasma membrane receptors determines their trafficking within target cells; and pathway(s) by which CT-A1 is translocated from the ER to the cytoplasm to reach its intracellular target and cause toxicity. During the next project period we will use a wide variety of novel methods from microbiology, genetics, biochemistry, cell biology and structural biology to investigate these important current issues concerning the structure and function of cholera toxin.