Mitochondrial Function in Bloodstream Trypanosoma brucei

布氏锥虫血流中的线粒体功能

• 批准号: 7617156
• 负责人: Marilyn Parsons
• 金额: $ 37.47万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7617156
• 关键词: Affinity; Affinity Chromatography; African Trypanosomiasis; Berenil; Biogenesis; Biological Assay; Biology; Blood Circulation; Bypass; Cell Nucleus; Cells; Cloning; Complement; Complex; Computer Simulation; Cytochromes; Disease; Drug Delivery Systems; Drug resistance; Gene Expression; Generations; Genes; Genetic; Goals; Hand; Leishmaniasis; Livestock; Mass Spectrum Analysis; Membrane Potentials; Mitochondria; Mitochondrial DNA; Monitor; Mutation; NADH dehydrogenase (ubiquinone); Nature; Organelles; Organism; Oxidative Phosphorylation; Parasites; Pathologic Processes; Pentamidine; Pharmaceutical Preparations; Play; Predisposition; Process; Protein Import; Proteins; RNA Editing; Research; Research Personnel; Resistance; Resistance development; Respiration; Role; Route; Staging; System; Technology; Testing; Toxic effect; Trypanosoma; Trypanosoma brucei brucei; Yeasts; base; cell growth; gel electrophoresis; genetic manipulation; health economics; in vivo; inhibitor/antagonist; insight; man; mitochondrial membrane; novel; pathogen; programs; protein purification; research study

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.

描述（由申请人提供）：锥虫是引起人类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产生而越来越无效。线粒体基因表达和功能对于寄生虫的生存至关重要——尽管在细长的血液（LS）形式中不存在氧化磷酸化——并且可能代表新药的强大靶标。然而，人们对 LS 阶段的线粒体生物学知之甚少，而且寄生虫细胞器的基本功能尚不清楚。该项目将测试以下假设：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，并且这些复合物中的一种或两种的基本成分通过线粒体表达。该假设基于这些复合物以 LS 形式活跃的证据以及编码这些复合物的假定亚基的线粒体基因的计算机识别。 (1) 通过沉默必需的核编码亚基的表达来灭活复合物 I 和 V，并检查对细胞生长和线粒体功能参数（如 ATP 水平、呼吸、膜电位和蛋白质输入）的影响。 (2) 通过鉴定体内基因产物的相互作用伙伴，将检验线粒体基因编码呼吸复合物 I 和 V 亚基的假设。蛋白质纯化和鉴定将通过亲和标记和质谱来实现。 (3) RNA 编辑的要求和 (1) 中确定的活性将在运动障碍 (dk) 锥虫中确定，这些锥虫已经失去了线粒体 DNA，因此可能表明潜在的耐药途径。 (4) 将通过关键核编码亚基的克隆和测序来鉴定 dk 锥虫中潜在的补偿性突变。这些研究将为锥虫的线粒体功能提供新的见解，验证新的药物靶标，并评估针对线粒体功能的药物的耐药性潜力。相关性：锥虫是单细胞寄生虫，是对健康和经济造成破坏性影响的疾病的病原体，例如非洲昏睡病和利什曼病。该项目将描述线粒体（一种细胞器）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。