STRUCTURE AND FUNCTION OF THE E. COLI STB ENTEROTOXIN

描述（改编自申请人摘要）：腹泻疾病 导致人类和家庭的严重发病率和死亡率 动物世界 大肠埃希氏菌（ETEC）的肠致病性菌株 是重要的代理商水性腹泻病，并诱发疾病， 多肽毒素的加工，其改变正常体液， 肠道电解质平衡 ETEC热稳定肠毒素（STs） 是通过诱导体液和电解质损失来介导体液和电解质损失的肽 肠道中的分泌途径。 两个ST（STa和STb）是 目前已确认。 STa是18-或19-mer酸性肽， 结合并激活鸟苷酸环化酶C（GCC），一种独特的肠道 膜鸟苷酸环化酶的形式。 cGMP的累积 通过激活CFTR氯化物导致氯化物分泌 频道 与STa相反，STb是48-mer碱性肽，其引起 在没有升高的环核苷酸的情况下的肠分泌， 另一个E.大肠杆菌分泌毒素。 工作在以前的 资助期间表明，STb导致肠道5- 羟色胺（5-羟色胺）和前列腺素E2的形成 （PGE 2），两种介质也已知参与霍乱毒素（CT） 介导的分泌。 然而，与CT不同，STb的细胞作用 引起百日咳毒素敏感的异源三聚体G Gi亚型蛋白。 在适当的实验条件下， STb介导的Gi激活引起胞浆钙离子升高 in treated处理cells细胞. STb还诱导G蛋白依赖性胞吐， 培养的肥大样细胞系导致体内释放5-HT。 最近的结构研究表明，STb是一个α-螺旋 两亲性肽，具有带正电荷的极性面， 螺旋和由第二个阿尔法螺旋形成的非极性面。 所述STb 结构和作用与一大类化合物一致 其在插入或穿透后直接激活G蛋白， 细胞膜 在本申请中，PI建议：1）确定 STb与靶细胞的特异性结合， 生物化学、生物物理学和超微结构技术; 2）确定 STb的细胞信号转导机制及G蛋白激活的作用 肠分泌; 3）研究肠道级联反应 STb作用引起的促分泌素及其在分泌中的特殊作用， 和4）确定负责细胞的结构特征， 和STb的分子作用。 阐明了细胞和 STb是一种独特的细菌毒素，其分子作用无疑将 不仅增加了我们对细菌肠毒素作用的理解， 此外，肠道维持正常液体的方式， 电解质平衡 其结果将是确定新的 肠分泌性疾病的治疗干预方法。

DESCRIPTION (Adapted from the applicant's abstract): Diarrheal diseases contribute to serious morbidity and mortality in humans and domestic animals world-wide. Enterotoxigenic strains of Escherichia coli (ETEC) are significant agents of watery diarrheal disease and induce illness by the elaboration of polypeptide toxins which alter the normal fluid and electrolyte balance in the gut. The ETEC heat-stable enterotoxins (STs) are peptides which mediate fluid and electrolyte loss by inducing hormone-like secretory pathways in the gut. Two STs (STa and STb) are currently recognized. STa is an 18- or 19-mer acidic peptide which binds to and activates guanylate cyclase C (GCC), a unique intestinal form of membrane guanylate cyclase. The resulting accumulation of cGMP results in chloride secretion via activation of the CFTR chloride channel. In contrast to STa, STb is a 48-mer basic peptide which causes intestinal secretion in the absence of elevated cyclic nucleotides, a hallmark of the other E. coli secretory toxins. Work in the previous funding period demonstrated that STb causes release of intestinal 5- hydroxytryptamine (serotonin, 5-HT) and formation of prostaglandin E2 (PGE2), two mediators also known to be involved in cholera toxin (CT) mediated secretion. Unlike CT, however, the cellular action of STb causes activation of a pertussis toxin-sensitive heterotrimeric G protein of the Gi subtype. Under appropriate experimental conditions, STb-mediated Gi activation causes an elevation of cytosolic calcium ions in treated cells. STb also induces G-protein dependent exocytosis from a cultured mast-like cell line resulting in release of 5-HT in vivo. Recent structural studies indicate that STb is an alpha-helical amphipathic peptide with a positively-charged polar face formed by one helix and a non-polar face formed by a second alpha helix. The STb structure and action are consistent with a broad group of compounds which directly activate G proteins following insertion or penetration of cell membranes. In this application the PI proposes to: 1) determine the specific association of STb with target cells by employing biochemical, biophysical and ultrastructural techniques; 2) determine the cellular signaling mechanism of STb and the role of G protein activation in intestinal secretion; 3) investigate the cascade of intestinal secretagogues evoked by STb action and their specific role in secretion, and 4) determine the structural features responsible for the cellular and molecular action of STb. The elucidation of the cellular and molecular action of STb, a unique bacterial toxin, will undoubtedly increase our understanding not only of bacterial enterotoxin action, but also, the means by which the intestine maintains normal fluid and electrolyte balance. The result will be the identification of new approaches to therapeutic intervention of intestinal secretory diseases.