Signaling GPI-phosphlipase C of a Trypanosome

描述（由申请人提供）：内吞作用和细胞信号传导对真核生物的生存力很重要。在布氏锥虫中，对细胞信号传导知之甚少，因为在模型真核生物中研究的主要受体在寄生虫基因组中无法识别。T.布氏杆菌具有糖基磷脂酰肌醇-磷脂酶C（GPI-PLC），其生物学功能尚不清楚。我们最近发现，GPI-PLC刺激转铁蛋白的内吞作用高达500%，并且这种生理效应需要酶活性。因此，我们假设GPI-PLC的裂解产物足以刺激内吞作用。与此一致的概念，外源性甘油二酯（DAG）促进内吞作用的寄生虫。因此，T.布氏杆菌具有调节胞吞作用的DAG受体。我们的长期目标是描绘的组成部分，并评估DAG信号在T。布鲁塞。为了实现这些目标，我们描述了用于鉴定DAG结合蛋白（TbDAGBP）的方法。在具体目标1中，我们将（i）克隆和表征从生物信息学分析预测的TbDAGBP;（ii）使用常规色谱法纯化TbDAGBP;和（iii）筛选TbDAGBP的cDNA表达文库。在特定目标2中，将通过敲低TbDAGBP的表达，并测试TbDAGBP缺陷型T.布鲁氏菌失去了响应DAG而刺激内吞作用能力。这些研究将导致发现新的DAG结合蛋白，因为在脊椎动物中用于识别DAG的C1结构域在T.布鲁塞。锥虫引起的疾病影响了全世界数百万人。本提案中描述的工作可能会导致发现新的信号通路，这些通路可能用于治疗人类非洲锥虫病。

DESCRIPTION (provided by applicant): Endocytosis and cell signaling are important for viability of eukaryotes. In Trypanosoma brucei, little is known about cell signaling because the major receptors studied in model eukaryotes are unrecognizable in the parasite genome. T. brucei has a glycosylphosphatidylinositol-phospholipase C (GPI-PLC) whose biological function(s) of GPI-PLC have been elusive. We found recently that GPI-PLC stimulates endocytosis of transferrin up to 500%, and that enzyme activity is needed for this physiological effect. Therefore, we hypothesized that a cleavage product of GPI-PLC would be sufficient to stimulate endocytosis. Consistent with this concept, exogenous diacylglycerol (DAG) promoted endocytosis in the parasite. Thus, T. brucei has receptors for DAG that regulate endocytosis. Our long-term goals are to delineate the components and evaluate the physiological significance of DAG signaling in T. brucei. Towards these goals, we describe approaches for identification of DAG-binding proteins (TbDAGBPs). In Specific Aim 1, we will (i) clone and characterize TbDAGBPs predicted from bioinformatic analysis; (ii) use conventional chromatography to purify TbDAGBPs; and (iii) screen a cDNA expression library for TbDAGBPs. In Specific Aim 2, effectors that link DAG signaling to the endocytic system will be identified, by knocking down expression of TbDAGBPs, and testing whether the TbDAGBP-deficient T. brucei lose ability to stimulate endocytosis in response to DAG. These studies will lead to discovery of novel DAG binding proteins because the C1-domain used in vertebrates for recognition of DAG is not detectable in the genome of T. brucei. Trypanosomes cause diseases that affect millions of people world-wide. Work described in this proposal may lead to discovery of new signaling pathways that may be targeted for treatment of human African trypanosomiasis.