Elucidating a G-protein signaling pathway in E. histolytica amoebic colitis

描述（由申请方提供）：由溶组织内阿米巴引起的阿米巴性结肠炎是一种主要的世界健康负担，估计每年导致100，000例死亡。感染性阿米巴滋养体对肠粘膜的侵袭依赖于由Rho家族GTP酶调控的动态细胞骨架。Siderovski实验室最近从E.溶组织菌基因组我自己的初步数据表明，EhRGS-RhoGEF以核苷酸依赖性方式结合EhG 1，并加速后者蛋白质的GT3活性。与哺乳动物中的G112/13通路类似，异源三聚体G蛋白信号通路可能调节Rho GT3活性，从而调节E.溶组织剂此外，G蛋白信号通路经常提供优良的药物靶标。 在特定目标1中，将通过鉴定EhRGS-RhoGEF的Rho GTdR底物来确定该G蛋白信号传导途径的分子组分。其激活的生化和结构机制将被阐明，分别使用核苷酸交换分析和X射线晶体学。特异性目的2将确定EhG 1和EhRGS-RhoGEF在阿米巴肠侵袭和破坏中的作用。拟议的实验将测试是否过度或不足的蛋白质的表达改变滋养体的能力，以帽表面受体，入侵细胞外基质，肠上皮细胞，或入侵感染小鼠的肠壁。 这些实验旨在阐明致病性重要的细胞信号级联在大肠杆菌。可以用于药物发现的溶组织菌。这项工作也将提供一个潜在的机制，阿米巴反应细胞外线索在肠道，进一步了解宿主-病原体的相互作用，在阿米巴结肠炎。拟议的培训和研究计划将提供必要的技能和知识基础，作为一个成功的职业生涯胃肠病医生，科学家。

DESCRIPTION (provided by applicant): Ameobic colitis, caused by Entamoeba histolytica, is a major world health burden, resulting in an estimated 100,000 deaths per year. Invasion of the intestinal mucosa by the infectious amoeba trophozoites depends on a dynamic cytoskeleton that is regulated by Rho family GTPases. The Siderovski lab has recently identified the first functional heterotrimeric G proteins (EhG123) and a regulator of G protein signaling (RGS) domain-containing Rho guanine nucleotide exchange factor (GEF) from the E. histolytica genome. My own preliminary data indicates that EhRGS-RhoGEF binds EhG1 in a nucleotide-dependent manner and accelerates the latter protein's GTPase activity. A heterotrimeric G protein signaling pathway, similar to the G112/13 pathway in mammals, likely regulates Rho GTPase activity and thus intestinal invasion by E. histolytica. Furthermore, G protein signaling pathways frequently provide excellent drug targets. In Specific Aim 1, the molecular components of this G protein signaling pathway will be determined by identifying a Rho GTPase substrate for EhRGS-RhoGEF. Biochemical and structural mechanisms for their activation wil be elucidated, using nucleotide exchange assays and X-ray crystallography, respectively. Specific Aim 2 will determine roles for EhG1 and EhRGS-RhoGEF in amoebic intestinal invasion and destruction. The proposed experiments will test whether over- or under-expression of either protein alters the ability of trophozoites to cap surface receptors, invade extracelular matrix, kil intestinal epithelial cels, or invade the intestinal walls of infected mice. These experiments are designed to elucidate a pathogenically important celular signaling cascade in E. histolytica that may be exploited for drug discovery. This work will also provide a potential mechanism for amoebic response to extracellular cues in the intestine, furthering our understanding of the host-pathogen interaction during amoebic colitis. The proposed training and research plans will provide the skills and knowledge base necessary for a successful career as a gastroenterologist physician-scientist.