Signaling GPI-phosphlipase C of a Trypanosome

描述(申请人提供)：内吞作用和细胞信号对真核生物的生存很重要。在布鲁氏锥虫中，人们对细胞信号转导知之甚少，因为在模式真核生物中研究的主要受体在寄生虫基因组中是无法识别的。布氏毛滴虫有一个糖基磷脂酰肌醇-磷脂酶C(GPI-PLC)，其生物学功能(S)一直难以捉摸。我们最近发现，GPI-PLC能刺激高达500%的转铁蛋白内吞作用，而这种生理作用需要酶的活性。因此，我们假设GPI-PLC的切割产物足以刺激内吞作用。与这一概念一致的是，外源二酰甘油(DAG)促进了寄生虫的内吞作用。因此，布氏毛滴虫有调节内吞作用的DAG受体。我们的长期目标是描述布鲁氏毛滴虫DAG信号的组成并评估其生理意义。为了实现这些目标，我们描述了鉴定DAG结合蛋白(TbDAGBPs)的方法。在具体目标1中，我们将(I)克隆和鉴定生物信息学分析预测的TbDAGBPs；(Ii)使用常规层析纯化TbDAGBPs；以及(Iii)筛选TbDAGBPs的cDNA表达文库。在具体目标2中，将通过下调TbDAGBPs的表达，并测试TbDAGBP缺陷的布氏毛滴虫是否失去刺激DAG反应的内吞作用的能力，来确定将DAG信号与内吞系统联系起来的效应器。这些研究将导致新的DAG结合蛋白的发现，因为在脊椎动物中用于识别DAG的C1结构域在布氏毛滴虫基因组中检测不到。锥虫引起的疾病影响着全世界数百万人。这项建议中描述的工作可能会导致发现新的信号通路，这些信号通路可能针对人类非洲锥虫病的治疗。

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.