CALCIUM PATHWAYS IN AFRICAN TRYPANOSOMES

非洲锥虫的钙途径

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/2062653
• 关键词: G protein; Trypanosoma; biological signal transduction; calcium channel; calcium flux; endoplasmic reticulum; fluorescent dye /probe; laboratory mouse; lysosomes; mitochondria; molecular cloning; pharmacology; receptor; triazoles

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（改编自申请人的摘要）：钙信号 途径被广泛用于触发细胞的快速变化 活动。钙信号被利用的程度 布氏锥虫协调复杂的生命周期事件，以及 药物干扰信号通路的可及性是 不太了解。由于以下原因，该领域的进展缓慢 缺乏合适的测试系​​统来监测信号转导 质膜和细胞内钙的释放 隔间。只有完整的细胞才能用于这些研究。 Ruben 博士的实验室利用系统方法来解决这个问题 问题。完整的锥虫负载有钙敏感的 荧光染料 Fura-2 用于监测细胞内游离钙浓度 (Ca2+)。该系统用于鉴定两亲性肽和 胺打开 G 蛋白偶联钙通道，并释放 储存钙。选择性打开锥虫的独特能力 然后利用钙通道来识别药剂 这扰乱了渠道活动。发现了两个新的对手。 本提案以这些结果为基础。具体目标 1 是 表征维持细胞器和能量依赖的蛋白质 钙稳态。钙稳态的持续破坏是 对真核细胞具有致命性。分离的细胞器将用于鉴定 哪些隔室能够钙转运以及机制 钙的流入和流出。重点放在溶酶体上， 内质网和线粒体。过度表达的完整细胞 内质网 Ca2+-ATP 酶将用于了解 该细胞器对体内钙稳态的贡献。这 钙敏感发光蛋白，水母发光蛋白将融合到 线粒体目标信号报告线粒体钙 完整的锥虫。具体目标 2 是表征分子 调节钙通道活性的两种蛋白质的水平特性； G蛋白和苄化三唑受体。这些的位置 细胞中的蛋白质，功能域的识别，以及 将确定药物结合的结构要求。具体目标 3、是评价锥虫钙通道的药理学。 苄基三唑破坏锥虫钙的活性 渠道。与有机化学家合作，放射性 这些化合物的衍生物将被合成，连同其 侧链含有不同的官能团。衍生品将 用于定量苄基化三唑受体数量和 亲和力：作为亲和标签，在过程中跟随受体蛋白 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。