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: 8124069
• 负责人: Dustin E Bosch
• 金额: $ 3.43万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8124069
• 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 Delivery Systems; 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. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Amoebic colitis and other complications arising from infection with the pathogen E. histolytica results in considerable morbidity and mortality worldwide, particularly in developing countries. We propose to study a new G-protein signaling pathway within E. histolytica and its relevance to pathogenicity and intestinal invasion. Like many analogous signaling pathways in humans, detailed structural and functional knowledge of a G-protein signaling pathway controlling E. histolytica pathogenicity is likely to greatly facilitate drug discovery.

描述(申请人提供)：阿米巴性结肠炎，由溶组织内阿米巴引起，是一个主要的世界卫生负担，估计每年导致10万人死亡。传染性阿米巴滋养体对肠粘膜的侵袭依赖于受Rho家族GTP酶调控的动态细胞骨架。西德罗夫斯基实验室最近从溶组棘球藻基因组中首次发现了具有功能的异三聚体G蛋白(EhG123)和含有G蛋白信号转导(RGS)结构域的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与EhG1结合，并加速后者的GTPase活性。一个异源三聚体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 GTP酶的活性，从而调节溶组性肠杆菌的肠道侵袭。此外，G蛋白信号通路经常提供极好的药物靶点。在特定目标1中，将通过鉴定EhRGS-Rhogef的Rho GTPase底物来确定这一G蛋白信号通路的分子组成。其激活的生化机制和结构机制将分别用核苷酸交换分析和X射线结晶学来阐明。具体目标2将确定EhG1和EhRGS-Rhogef在阿米巴肠道侵袭和破坏中的作用。拟议中的实验将测试这两种蛋白的过度表达或表达不足是否会改变滋养体覆盖表面受体的能力，入侵细胞外基质，杀死肠道上皮细胞，或入侵受感染小鼠的肠壁。这些实验旨在阐明在溶组织埃希氏菌中一个重要的细胞信号级联反应，该信号可能被用于药物开发。这项工作还将为阿米巴对肠道细胞外线索的反应提供一个潜在的机制，进一步加深我们对阿米巴结肠炎过程中宿主-病原体相互作用的理解。拟议的培训和研究计划将提供作为胃肠病专家、内科医生和科学家的成功职业生涯所需的技能和知识基础。 与公共卫生的相关性：项目叙述阿米巴结肠炎和其他因感染溶组织性肠杆菌而引起的并发症在世界范围内造成相当大的发病率和死亡率，特别是在发展中国家。我们建议研究一种新的G蛋白信号通路及其与致病性和肠道侵袭的关系。与人类许多类似的信号通路一样，对控制溶组织性肠杆菌致病性的G蛋白信号通路的详细结构和功能了解可能会极大地促进药物的发现。