Structure and function of the Trypanosome TIM complex

锥虫 TIM 复合物的结构和功能

• 批准号: 10087686
• 负责人: Minu Chaudhuri
• 金额: $ 3.01万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-22 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10087686
• 关键词: Affect; Affinity Chromatography; Antibodies; Binding Proteins; Biological Assay; Blood Circulation; Cell Line; Cell Nucleus; Cell physiology; Cells; Centrifugation; Characteristics; Complex; Cytosol; DNA; Data; Development; Disease; Epitopes; Eukaryota; Foundations; Gel; Glycerol; Goals; Human; In Vitro; Insect Vectors; Knowledge; Laboratories; Leishmania; Mammals; Mass Spectrum Analysis; Membrane; Mitochondria; Mitochondrial Proteins; Molecular Sieve Chromatography; Mutation; N-terminal; Nuclear; Organelles; Parasites; Plants; Property; Protein Import; Proteins; Protozoa; Role; Signal Transduction; Site-Directed Mutagenesis; Source; Structure; Substrate Specificity; System; Techniques; Testing; Transfer RNA; Trypanosoma; Trypanosoma brucei brucei; base; design; fungus; genetic approach; in vivo; insight; knock-down; mutant; novel; novel therapeutics; overexpression; protein complex; protein protein interaction; reverse genetics; tool; translocase; two-dimensional

Project Summary: The majority of proteins essential for mitochondrial functions are encoded in the nuclear DNA and subsequently imported from the cytosol by translocases of the mitochondrial outer and inner membranes, termed TOM and TIM, respectively. These translocases are multimeric protein complexes, which are extensively studied in fungi and later in humans and plants. However, mitochondrial protein import machinery has been poorly characterized in trypanosomatids, a group of ancient and early branching unicellular eukaryotes that possesses a single mitochondrion per cell. Recent studies by our laboratory and others identified a number of non-canonical protein components for this essential cellular process in Trypanosoma brucei. As part of this machinery, we previously characterized TbTim17, which is critical for mitochondrial protein import and thus essential for parasite survival in two major developmental forms, the procyclic and bloodstream forms, found in the insect vector and the mammalian bloodstream, respectively. TbTim17 is present in multiple protein complexes within the range of 300-1100 kDa. Using pull down assays we identified one relatively conserved protein, Tim50, and several other novel proteins. Among these, we found TbTim62, a non-canonical Tim protein, act as a critical assembly factor of the TbTim17 protein complexes and have a major effect on TbTim17 stability. In addition, we found that Tim50 interacts with TbTim17 and is involved in the import of N-terminal signal containing mitochondrial proteins. Besides, TbTim17 also associates with a group soluble small TbTims. In contrast to the two TIM complexes (TIM23 and TIM22) with different substrate specificities in fungi to mammals, trypanosomatids most likely possess a single TIM (TbTIM17) that is capable of importing various types of mitochondrial proteins. Based on these results we hypothesized that TbTim17 forms an evolutionarily divergent and modular type TIM complex by association with novel TbTims and imports different types of substrate protein into mitochondria. We will test this hypothesis by three aims; 1) to define the structural components of the TbTim17 protein complex; 2) to determine the unique and interactive functions of TbTim17, TbTim62, TbTim50, TbTim54, and small TbTims in T. brucei, and 3) to determine the structural domains of TbTims critical for their functions. We will use many conventional and specialized techniques to evaluate the properties of these TbTims, including their substrate specificities, ability to assemble into the TIM complex, role in the stability of this complex, protein-protein interactions, and unique structure-function relations. Together, these studies will elucidate the subunit composition and function of the divergent TbTIM complex in T. brucei that can also be extrapolated for other trypanosomatid parasites and thus illuminate the mechanism of mitochondrial protein import in a group of the earliest eukaryotes.

项目摘要： 线粒体功能必不可少的大多数蛋白质都编码在核DNA和 随后通过线粒体外膜和内膜的易位酶从细胞质进口， 分别称汤姆和蒂姆。这些易位酶是多聚体蛋白质复合物， 在真菌中进行了广泛的研究，后来在人类和植物中进行了研究。但是，线粒体蛋白进口机械 在锥虫中的特征很差，这是一群古老和早期分支的单细胞 每个细胞具有单个线粒体的真核生物。我们的实验室和其他人的最新研究 确定了锥虫瘤中这种必需细胞过程的许多非典型蛋白成分 布鲁西。作为此机械的一部分，我们先前表征了TBTIM17，这对于线粒体至关重要 蛋白质导入，因此对于两种主要发育形式的寄生虫生存至关重要，Procyclic和 血液形成，分别在昆虫载体和哺乳动物的血液中发现。 TBTIM17是 存在于300-1100 kDa范围内的多个蛋白质复合物中。使用我们确定的下拉测定法 一种相对保守的蛋白质，TIM50和其他几种新型蛋白质。其中，我们发现了TBTIM62，a 非典型的TIM蛋白，充当TBTIM17蛋白复合物的关键装配因子，并具有A 对TBTIM17稳定性的主要影响。此外，我们发现TIM50与TBTIM17相互作用，并参与 含有线粒体蛋白的N末端信号的进口。此外，TBTIM17还与 组可溶性小tbtims。与具有不同底物的两个TIM复合物（TIM23和TIM22）相反 真菌对哺乳动物的特异性很可能具有能够具有单一的TIM（TBTIM17） 导入各种类型的线粒体蛋白。基于这些结果，我们假设TBTIM17 通过与新型的TBTIM和进口相关联，形成进化上不同的模块化型TIM复合物 不同类型的底物蛋白进入线粒体。我们将以三个目标检验这一假设。 1）定义 TBTIM17蛋白复合物的结构成分； 2）确定 TBTIM17，TBTIM62，TBTIM50，TBTIM54和T. Brucei中的小tbtims和3）确定结构性 TBTIMS的域对其功能至关重要。我们将使用许多常规和专业技术来 评估这些TBTIM的属性，包括其底物特异性，可以组装到TIM 复杂的，在这种复合物的稳定性中的作用，蛋白质 - 蛋白质相互作用以及独特的结构功能 关系。总之，这些研究将阐明发散TBTIM的亚基组成和功能 布鲁氏菌中的复合物，也可以针对其他锥形寄生虫外推，从而照亮 线粒体蛋白在最早的真核生物中的进口机理。

项目成果

Erratum for Tripathi et al., "The Cross Talk between TbTim50 and PIP39, Two Aspartate-Based Protein Phosphatases, Maintains Cellular Homeostasis in Trypanosoma brucei".

Tripathi 等人的勘误表，“TbTim50 和 PIP39（两种基于天冬氨酸的蛋白磷酸酶）之间的串扰维持布氏锥虫的细胞稳态”。

• DOI: 10.1128/msphere.00607-19
• 发表时间: 2019
• 期刊: mSphere
• 影响因子: 4.8
• 作者: Tripathi,Anuj;Singha,UjjalK;Paromov,Victor;Hill,Salisha;Pratap,Siddharth;Rose,Kristie;Chaudhuri,Minu
• 通讯作者: Chaudhuri,Minu