CALCIUM PATHWAYS IN AFRICAN TRYPANOSOMES

非洲锥虫的钙途径

基本信息

批准号：
2062653
负责人：
Lawrence Ruben
金额：
$ 16.65万
依托单位：
SOUTHERN METHODIST UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-03-01 至 1997-11-30
项目状态：
已结题

项目摘要

DESCRIPTION (Adapted from the Applicant's Abstract): Calcium signal pathways are used extensively to trigger rapid changes in cellular activity. The extent to which calcium signals are utilized by Trypanosoma brucei to coordinate complex life cycle events, and the accessibility of the signal pathways to pharmaceutical disruption is poorly understood. Progress in this area has been slow due to the absence of a suitable test system to monitor signal transduction across the plasma membrane and release of calcium from intracellular compartments. Only intact cells can be used for these studies. Dr. Ruben's laboratory utilized a systematic approach to solve this problem. Intact trypanosomes were loaded with the calcium sensitive fluorochrome Fura-2 to monitor intracellular free calcium concentrations (Ca2+). This system was used to identify amphipathic peptides and amines that opened a G protein- coupled calcium channel, and released stored calcium. The unique ability to selectively open trypanosome calcium channels was then exploited to identify pharmaceutical agents that disrupted channel activity. Two novel antagonist were discovered. The present proposal builds upon these results. Specific Aim 1, is to characterize the organelles and energy dependent proteins that maintain calcium homeostasis. Sustained disruption of calcium homeostasis is lethal to eukaryotic cells. Isolated organelles will be used to identify which compartments are capable of calcium transport, and the mechanisms of calcium influx and eflux. Emphasis is placed upon the lysosomes, endoplasmic reticulum and mitochondria. Intact cells that over-express the endoplasmic reticulum Ca2+-ATPase will be used to understand the contribution of this organelle to calcium homeostasis in vivo. The calcium-sensitive luminescent protein, aequorin will be fused to a mitochondrial target signal to report on mitochondrial calcium in the intact trypanosome. Specific Aim 2, is to characterize at the molecular level properties of two proteins that modulate calcium channel activity; the G protein, and benzylated triazole receptor. The location of these proteins in the cell, the identification of functional domains, and structural requirement for drug binding will be determined. Specific Aim 3, is to evaluate the pharmacology of the trypanosome calcium channels. Benzylated triazole disrupt the activity of trypanosome calcium channels. In collaboration with an organic chemist, radioactive derivatives of these compounds will be synthesized, along with whose side chains contain different functional groups. The derivatives will be used to quantitate the benzylated triazole receptor number and affinity: as an affinity tag to follow the receptor protein during purification; and to evaluate side chain requirements for drug interactions with the receptor. The effects of these drugs on acute and chronic infections in mice will be determined.

描述（根据申请人的摘要改编）：钙信号途径被广泛用于触发细胞的快速变化活动。钙信号的使用程度布鲁氏锥虫以协调复杂的生命周期事件，并信号途径到药物破坏的可及性是理解不佳。由于该领域的进展一直很慢没有合适的测试系统来监视跨越信号的转导质膜和细胞内钙的释放车厢。只有完整的细胞才能用于这些研究。鲁本博士的实验室利用了一种系统的方法来解决这个问题问题。完整的锥虫加载了钙敏感的荧光色素fura-2监测细胞内游离钙浓度（Ca2+）。该系统用于识别两亲性肽和打开G蛋白偶联钙通道并释放的胺储存的钙。选择性打开锥虫体的独特能力然后利用钙通道以识别药物这种破坏的通道活动。发现了两个新颖的对手。目前的提案基于这些结果。特定目标1是表征维持的细胞器和依赖能量的蛋白质钙稳态。钙稳态的持续破坏是致命的真核细胞。孤立的细胞器将用于识别哪些隔室能够钙传输和机制钙的流入和eflux。重点放在溶酶体上，内质网和线粒体。过表达的完整细胞内质网Ca2+-ATPase将用于了解该细胞器对体内钙稳态的贡献。这钙敏感的发光蛋白，aequorin将融合到线粒体靶信号报告有关线粒体钙完整的锥体。特定的目标2是在分子上表征调节钙通道活性的两种蛋白质的水平特性； G蛋白和苄基三唑受体。这些位置细胞中的蛋白质，功能结构域的鉴定和将确定药物结合的结构要求。具体目标 3，是为了评估锥虫钙通道的药理学。苄基三唑破坏锥虫钙的活性频道。与有机化学家合作，放射性这些化合物的衍生物将与之合成侧链包含不同的官能团。衍生物会用于定量偶联的三唑受体数量和亲和力：作为在遵循受体蛋白的亲和力标签纯化;并评估药物的侧链要求与受体的相互作用。这些药物对急性和将确定小鼠的慢性感染。