STRUCTURE AND FUNCTION OF THE E. COLI STB ENTEROTOXIN

描述(改编自申请人的摘要)：腹泻病 在人类和家庭中造成严重的发病率和死亡率 世界各地的动物。产肠毒素大肠杆菌(ETEC) 是水样腹泻病的重要病原体，并通过 改变正常体液的多肽毒素的研究进展 肠道内的电解质平衡。ETEC耐热肠毒素(STS) 是通过诱导体液和电解质流失的多肽 肠道中类似荷尔蒙的分泌途径。两个STS(STA和STB)是 目前已被认可。StA是一种18或19聚体的酸性多肽 结合并激活鸟苷环化酶C(GCC)，一种独特的肠道 膜鸟苷环化酶的形式。由此产生的cGMP积累 通过激活CFTR氯离子导致氯离子分泌 频道。与STA不同，STB是一种由48个氨基酸组成的碱性多肽，可引起 没有升高的环核苷酸的肠道分泌物，a 这是其他大肠杆菌分泌毒素的特征。在上一版本中工作 资助期显示，STB导致肠道5-羟色胺的释放 羟色胺与前列腺素E_2的形成 (PGE2)，两个已知与霍乱毒素(CT)有关的介体 中介分泌。然而，与CT不同，STB的细胞作用 引起百日咳毒素敏感的异三聚体G的激活 Gi亚型的蛋白质。在适当的实验条件下， STB介导的胃肠道激活引起细胞内钙离子升高 在处理过的细胞中。STB还可诱导G蛋白依赖性胞吐作用 体内能释放5-羟色胺的肥大样细胞系。 最近的结构研究表明，STB是一个α-螺旋 一种带正电极面的两亲性多肽 螺旋和由第二个阿尔法螺旋形成的非极面。机顶盒 结构和作用与一大类化合物一致 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与靶细胞的特异性关联 生化、生物物理和超微结构技术；2)测定 STB的细胞信号机制及G蛋白激活的作用 在肠道分泌物中；3)研究肠道的级联 STB作用引起的促分泌剂及其在分泌中的特殊作用 和4)确定负责细胞的结构特征 STB的分子作用。细胞和细胞因子的阐明 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道毒素作用的了解，而且 此外，肠道维持正常液体和 电解质平衡。其结果将是识别新的 肠道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.