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

阐明溶组织阿米巴结肠炎中的 G 蛋白信号通路

• 批准号: 8466965
• 负责人: Dustin E Bosch
• 金额: $ 3.57万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8466965
• 关键词: Actins; Amebiasis; Amebic colitis; Amoeba genus; Binding; Biochemical; Biological Assay; Cell surface; Cessation of life; Chemotaxis; Colitis; Complement; Cues; Cytoskeleton; Data; Developing Countries; Development; Drug Targeting; Entamoeba histolytica; Epithelial; Event; Extracellular Matrix; Family; G-Protein Signaling Pathway; GTP-Binding Protein Regulators; GTP-Binding Proteins; Gastroenterologist; Genome; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric G Protein Subunit; Heterotrimeric GTP-Binding Proteins; Homologous Gene; Human; Infection; Intestinal Mucosa; Intestines; Invaded; Knowledge; Laboratories; Link; Mammals; Mediating; Modeling; Molecular; Monitor; Morbidity - disease rate; Mus; Nucleotides; Nutrient; Pathogenesis; Pathogenicity; Pathway interactions; Physicians; Play; Process; Proteins; RGS Domain; Receptor Aggregation; Research Training; Role; Scientist; Signal Pathway; Signal Transduction; Stimulus; Surface; System; Testing; Work; World Health; X-Ray Crystallography; base; career; cell killing; design; drug discovery; extracellular; genome sequencing; in vivo; indexing; knowledge base; leukemia; mortality; novel therapeutic intervention; overexpression; pathogen; receptor; research study; response; rho; rho GTP-Binding Proteins; rho guanine nucleotide exchange factor p115; skills; success

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.

描述（由申请人提供）：由溶组织内阿米巴引起的阿米巴性结肠炎是世界上一个主要的健康负担，估计每年造成10万人死亡。传染性阿米巴滋养体对肠黏膜的侵袭依赖于由Rho家族gtp酶调节的动态细胞骨架。Siderovski实验室最近发现了首个功能性异三聚体G蛋白（EhG123）和G蛋白信号传导（RGS）结构域调节剂，该结构域含有Rho鸟嘌呤核苷酸交换因子（GEF）。我自己的初步数据表明，EhRGS-RhoGEF以核苷酸依赖的方式结合EhG1，并加速后者蛋白的GTPase活性。异三聚体G蛋白信号通路，类似于哺乳动物中的G112/13通路，可能调节Rho GTPase活性，从而调节溶组织芽胞杆菌的肠道入侵。此外，G蛋白信号通路经常提供良好的药物靶点。在Specific Aim 1中，G蛋白信号通路的分子组成将通过鉴定EhRGS-RhoGEF的Rho GTPase底物来确定。它们激活的生化和结构机制将分别通过核苷酸交换测定和x射线晶体学来阐明。特异性Aim 2将确定EhG1和EhRGS-RhoGEF在阿米巴肠道侵袭和破坏中的作用。该实验将测试这两种蛋白的过表达或过低表达是否会改变滋养体覆盖表面受体、侵入细胞外基质、杀死肠上皮细胞或侵入感染小鼠肠壁的能力。这些实验旨在阐明溶组织芽胞杆菌中一个重要的致病细胞信号级联，可能用于药物发现。这项工作还将为肠道内阿米巴对细胞外信号的反应提供潜在的机制，进一步加深我们对阿米巴结肠炎期间宿主-病原体相互作用的理解。拟议的培训和研究计划将为胃肠病学医师和科学家的成功职业生涯提供必要的技能和知识基础。