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Signaling GPI-phosphlipase C of a Trypanosome

锥虫的 GPI-磷脂酶 C 信号传导

基本信息

批准号：
8072926
负责人：
KOJO A. MENSA-WILMOT
金额：
$ 1.72万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-24 至 2010-08-15
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：内吞作用和细胞信号传导对于真核生物的生存能力很重要。在布氏锥虫中，人们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。布氏锥虫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 可刺激转铁蛋白的内吞作用高达 500%，而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。与这一概念一致，外源性二酰甘油（DAG）促进了寄生虫的内吞作用。因此，布氏锥虫具有调节内吞作用的 DAG 受体。我们的长期目标是描绘布氏锥虫中 DAG 信号传导的组成部分并评估其生理意义。为了实现这些目标，我们描述了鉴定 DAG 结合蛋白 (TbDAGBP) 的方法。在具体目标 1 中，我们将 (i) 克隆并表征通过生物信息分析预测的 TbDAGBP； (ii) 使用常规色谱法纯化 TbDAGBP； (iii) 筛选 TbDAGBP 的 cDNA 表达文库。在具体目标 2 中，将通过敲低 TbDAGBP 的表达来鉴定将 DAG 信号传导与内吞系统联系起来的效应子，并测试 TbDAGBP 缺陷的布氏锥虫是否失去响应 DAG 刺激内吞作用的能力。这些研究将导致新型 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 T. brucei 的基因组中无法检测到。锥虫引起的疾病影响了全世界数百万人。该提案中描述的工作可能会发现新的信号通路，这些信号通路可能是治疗人类非洲锥虫病的目标。