Mitochondrial inner membrane protein translocase in Trypanosoma brucei

布氏锥虫线粒体内膜蛋白转位酶

基本信息

批准号：
8525404
负责人：
Minu Chaudhuri
金额：
$ 31.59万
依托单位：
MEHARRY MEDICAL COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8525404
关键词：
African Trypanosomiasis Biogenesis Biological Assay Blood Blood Circulation Cell Survival Characteristics Co-Immunoprecipitations Complex Cytosol Data Development Disease Domestic Animals Drug Targeting Eukaryota Fluorescence Resonance Energy Transfer Genes Genome Goals Homologous Gene Homologous Protein Human In Vitro Infectious Agent Inner mitochondrial membrane Insect Vectors Knock-out Laboratories Life Life Cycle Stages Link Mammals Maps Mediating Membrane Membrane Proteins Metabolic Mitochondria Mitochondrial Matrix Mitochondrial Proteins Molecular Mutate Mutation N-terminal Nuclear Organelles Organism Outer Mitochondrial Membrane Parasites Pharmaceutical Preparations Phosphoric Monoester Hydrolases Play Process Production Protein Import Proteins Proteome Protozoa RNA Interference Regulation Role Signal Transduction Specificity Structure Substrate Specificity Techniques Testing Trypanosoma Trypanosoma brucei brucei Variant Voltage-Dependent Anion Channel Yeasts base cell growth design fungus genome-wide hemoflagellate in vivo insight molecular mass mutant new therapeutic target novel parasite genome protein complex protein protein interaction therapeutic target translocase yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Similar to other eukaryotes, the majority of mitochondrial proteins in Trypanosoma brucei, the infectious agent of a fatal disease (African trypanosomiasis) in human and domestic animals, are nuclear-encoded and need to be imported into mitochondria. However, the protein import machinery remains elusive in this organism. Fungi and higher eukaryotes possess three homologous proteins Tim17, Tim23, and Tim22, which formed two translocases (TIMs) in the inner membranes of mitochondria that have distinct specificities for different types of signal bearing proteins. In contrast, T. brucei possesses a single homolog of these proteins, TbTim17, which we found is present in a larger molecular mass protein complex in the mitochondrial inner membrane and is required for the import of both N-terminal and internal targeting signal containing proteins into mitochondria. Thus T. brucei may possess a single TIM complex capable of importing multiple types of matrix and inner membrane proteins. How a single protein in T. brucei performs functions of three different proteins in other eukaryotes remains enigmatic. We also have identified a Tim17- interacting protein (TbTim50) and genome searches indicate that there are other Tim50 related proteins in T. brucei. All of these variants possess a characteristic phosphatase domain. In addition to its role in mitochondrial protein import and cell growth, we found that TbTim50 over expression reduces mitochondrial ATP production via regulation of the expression level of the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. Together, this suggests that mitochondrial protein importer molecules such as Tim50 may be linked to other activities of this organelle besides importing proteins into mitochondrial matrix and inner membrane. In this project we will characterize the function of Tim50 and Tim50-related proteins in T. brucei and identify the Tim17 and Tim50 interacting domain(s) crucial for their function. We will also investigate how Tim50 is connected to VDAC expression, which is connected to regulation of mitochondrial metabolic activities. We will use various gene manipulation techniques, in vitro and in vivo protein import assays, FRET analysis, yeast two-hybrid analysis, and expression of TbTim50 mutants to study the function and protein/protein interactions of Tim50 and Tim17 in T. brucei. Characterization of the novel structure and function of T. brucei Tims may provide insights that can be utilized to define a new chemotherapeutic target for African trypanosomiasis.

描述（由申请人提供）：与其他真核生物类似，Brucei锥虫瘤中的大多数线粒体蛋白是人类和家养动物的致命疾病（非洲锥虫病）的感染剂，是核编码的，需要进口到线粒体中。但是，在这种生物体中，蛋白质进口机器仍然难以捉摸。真菌和较高的真核生物具有三种同源蛋白Tim17，Tim23和Tim22，它们在线粒体的内膜中形成了两个易位酶（TIM），它们对于不同类型的信号轴承蛋白具有不同的特异性。相比之下，布雷西（T. brucei）具有这些蛋白质TBTIM17的单个同源物，我们发现它存在于线粒体内膜中的较大的分子质量蛋白复合物中，并且是将含有N-末端和内部靶向信号含有蛋白的N-末端和内部靶向信号的进口所必需的。因此，布鲁氏菌可能具有能够导入多种类型的基质和内膜蛋白的单一TIM复合物。布鲁氏菌中的单个蛋白如何在其他真核生物中执行三种不同蛋白的功能仍然神秘。我们还确定了TIM17相互作用的蛋白质（TBTIM50），基因组搜索表明T. Brucei中还有其他TIM50相关的蛋白质。所有这些变体都具有特征性的磷酸酶结构域。除了其在线粒体蛋白进口和细胞生长中的作用外，我们还发现，TBTIM50通过调节线粒体外膜上电压依赖性阴离子通道（VDAC）的表达水平来减少线粒体ATP的产生。总之，这表明线粒体蛋白导入的分子（例如Tim50）可能与该细胞器的其他活性有关，除了将蛋白质导入线粒体基质和内膜。在此项目中，我们将表征T. Brucei中TIM50和TIM50相关蛋白的功能，并确定TIM17和TIM50相互作用域对其功能至关重要。我们还将研究TIM50如何连接到VDAC表达，这与线粒体代谢活性的调节有关。我们将使用各种基因操纵技术，体外和体内蛋白进口测定，FRET分析，TBTIM50突变体的表达来研究TIM50和TIM17在T. brucei中的功能和蛋白/蛋白质相互作用。 t. brucei tims的新结构和功能的表征可以提供可用于定义非洲锥虫病的新化学治疗靶标的见解。