Genetic Analysis of Cholera Toxin Structure and Function

描述(改编自申请者摘要)：约15亿 腹泻病例每年导致400万5岁以下儿童死亡 陈年霍乱和500万至700万霍乱病例造成约10万人死亡。霍乱毒素 霍乱弧菌(CT)引起霍乱大量水样腹泻。 产肠毒素大肠杆菌(ETEC)在中国引起高达20%的腹泻疾病 ，并产生称为LTI和LTII的不耐热肠毒素 在结构和功能上与CT密切相关的基因。最好的电流 霍乱疫苗只能在短期内提供适度的保护 时间和在美国没有许可证，也没有疫苗 人类用来对抗ETEC。CT和相关肠毒素是有效的免疫原和 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 神经节苷脂在内吞和囊泡运输中的作用、定位和/或 消融神经通路，以及许多其他细胞功能。我们研究了 CT的结构和功能，并使用LTI和LTII进行比较研究 探索它们之间功能差异的分子基础。我们的龙 学期目标是阐明CT生物学活性的分子基础 和相关的肠毒素，并利用这些知识来设计小说 预防或治疗肠道中毒的基于结构的疫苗和治疗方法 腹泻。CT、LTI或LTII作为疫苗成分也被广泛研究， 预防或治疗无关疾病的佐剂或免疫调节剂 肠毒性腹泻。CT结构和功能的几个重要问题 尚不了解的内容包括识别和描述： 活化CT-A1催化能力的构象变化 CT全毒素的划痕和还原；CT-A1上决定其活性的基序 与Gsalpha/beta/Gamma作为ADP核糖化底物的相互作用 以ADP-核糖化因子(ARF)作为催化活性的刺激物； CT-A和CT-B能够自发组装成CT的特征 全毒素；肠毒素与质膜结合的机制 受体决定其在靶细胞内的转运；而通路(S)通过 哪种CT-A1从内质网转移到细胞质以到达其 细胞内靶标并引起毒性。在下一个项目期间，我们将 使用从微生物学、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.