Mitochondrial Function in Bloodstream Trypanosoma brucei

描述（由申请方提供）：锥虫是引起人和牲畜疾病的原生动物病原体。大多数药物由于毒性和耐药性的发展而越来越无效。线粒体基因表达和功能对于寄生虫的生存至关重要-尽管在细长血流（LS）形式中缺乏氧化磷酸化-并且可能代表新药的强大靶标。然而，LS阶段的线粒体生物学知之甚少，寄生虫的细胞器的基本功能也不清楚。该项目将测试呼吸复合物I（NADH：泛醌氧化还原酶）和/或V（ATP合酶）是LS寄生虫生存所需的假设，并且这些复合物中的一种或两种的基本成分都是通过呼吸表达的。这一假设是基于证据表明，这些复合物是活跃的LS形式和在电脑识别的线粒体基因编码这些复合物的推定亚基。(1)复合物I和V将通过沉默必需的核编码亚基的表达而失活，并将检查对细胞生长和对线粒体功能参数（如ATP水平、呼吸、膜电位和蛋白质输入）的影响。(2)假设将被测试，线粒体基因编码呼吸复合物I和V的亚基，通过确定在体内的基因产物的相互作用的合作伙伴。将通过亲和标记和质谱法实现蛋白质纯化和鉴定。(3)将在运动发育障碍（dk）锥虫中确定（1）中确定的RNA编辑和活性需求，这些锥虫已丢失线粒体DNA，因此可能表明潜在的耐药性途径。(4)dk锥虫中潜在的补偿突变将通过关键的核编码亚基的克隆和测序来鉴定。这些研究将为锥虫的线粒体功能提供新的见解，验证新的药物靶点，并评估对靶向线粒体功能的药物的耐药性。相关性：锥虫是单细胞寄生虫，是非洲昏睡病和利什曼病等具有毁灭性健康和经济后果的疾病的病原体。该项目将描述寄生虫-细胞器-在寄生虫致病阶段的作用。这项研究将有助于确定新的药物靶点，并了解现有药物的作用。

DESCRIPTION (provided by applicant): Trypanosomatids are protozoan pathogens that cause disease in man and livestock. Most drugs are increasingly ineffective due to toxicity and development of resistance. Mitochondrial gene expression and function are essential for survival of the parasite - despite the absence of oxidative phosphorylation in the long slender bloodstream (LS) form - and may represent powerful targets for new drugs. However, mitochondrial biology in the LS stage is poorly understood and the essential function of the organelle for the parasite is not known. This project will test the hypothesis that respiratory complexes I (NADH:ubiquinone oxidoreductase) and/or V (ATP synthase) are required for viability of LS parasites and that essential components of one or both of these complexes are mitochondrially expressed. This hypothesis is based on evidence that these complexes are active in the LS form and on the in silico identification of mitochondrial genes encoding putative subunits of these complexes. (1) Complexes I and V will be inactivated by silencing the expression of essential nuclearly encoded subunits and the effects on cell growth and on parameters of mitochondrial function such as ATP levels, respiration, membrane potential, and protein import will be examined. (2) The hypothesis will be tested that mitochondrial genes encode subunits of respiratory complexes I and V by identifying the interaction partners of the gene products in vivo. Protein purification and identification will be achieved through affinity tagging and mass spectrometry. (3) The requirement for RNA editing and activities identified in (1) will be determined in dyskinetoplastic (dk) trypanosomes, which have lost their mitochondrial DNA and may thus indicate potential routes for drug resistance. (4) Potential compensatory mutations in dk trypanosomes will be identified by cloning and sequencing of key nuclearly encoded subunits. These studies will provide novel insights into mitochondrial function in trypanosomatids, validate new drug targets, and assess the potential for resistance to drugs targeting mitochondrial function. Relevance: Trypanosomatids are unicellular parasites and the causative agents of diseases with devastating health and economic consequences, such as African sleeping sickness and Leishmaniasis. This project will characterize the role of the mitochondrion - a cell organelle - in the disease-causing stage of the parasite. This research will help to identify new drug targets and to understand the action of existing drugs.