Characterization of Trypanosome telomere complex

锥虫端粒复合物的表征

• 批准号: 7211023
• 负责人: Bibo Li
• 金额: $ 29.95万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7211023
• 关键词: Africa South of the Sahara; African Trypanosomiasis; Alleles; Antigenic Variation; Binding; Cattle; Cell Line; Cells; Chromosomes; Cloning; Co-Immunoprecipitations; Complex; Genetic Transcription; Goals; Human; Hybrids; Immune system; Knock-in Mouse; Knock-out; Mass Spectrum Analysis; Membrane Glycoproteins; Monoclonal Antibodies; Mutagenesis; Mutation; Parasites; Pathogenesis; Phenotype; Play; Point Mutation; Polymerase Chain Reaction; Protein Overexpression; Proteins; RNA Interference; Role; Structure; Trypanosoma; Trypanosoma brucei brucei; Yeasts; cDNA Library; in vivo; mutant; nagana; pathogen; telomere

DESCRIPTION (provided by applicant): T. brucei is a parasite that causes sleeping sickness in humans and Nagana in cattle in sub-Saharan Africa. When growing in mammalian host, T. brucei cells regularly switch their major surface glycoprotein, to evade the host immune system - a phenomenon called antigenic variation. These surface glycoproteins are exclusively expressed from one of ~ 20 loci located next to chromosome ends - telomeres. Telomeres in yeasts form a specialized structure that influences transcription of genes located close by. A similar phenomenon has also been observed in T. brucei, and telomeres may play an important role in antigenic variation. The ultimate goal of telomere studies in Trypanosoma brucei is to understand telomere functions in antigenic variation, an essential aspect of T. brucei pathogenesis. The fundamental step would be to identify telomere components and characterize their functions, which is the primary goal of this proposal. Specific Aim 1: To purify tbTRF (a double-stranded telomere DMA binding factor in T. brucei) protein complex, using two approaches: 1) Sequential immunoprecipitate pull-down of FLAG-HA-HA tagged tbTRF using anti-FLAG and anti-HA monoclonal antibodies. Proteins co-immunoprecipitated will be identified by mass spectrometry. 2) Yeast 2-hybrid screen using lexA-tbTRF as bait and a T. brucei GAD-fusion cDNA library. Candidates for tbTRF-interaction factors will be first confirmed for their interaction with tbTRF in vivo by co-immunoprecipitation. Positive clones will be characterized for their roles in antigenic variation and telomere functions by examination of phenotypes in knockout or RNAi knockdown cell lines. Specific Aim 2: Select non-lethal interaction-deficient tbTRF mutations in the tbTRFH domain to better characterize tbTRF's function. Random point mutations or small deletions will be generated in the tbTRFH domain, using PCR cloning. Mutants that lose the interaction with wild-type tbTRF but remain the ability to interact with them will be screened using both conventional and reverse 2-hybrid analysis. Cell lines with knock-in or overexpression of these mutant alleles will be characterized for their phenotypes in antigenic variation. The identification of telomere complex components and subsequent characterization of their functions would be a good starting point for studying telomere functions in antigenic variation, an essential aspect of T. brucei pathogen.

描述（由申请人提供）：布氏锥虫是一种寄生虫，可导致撒哈拉以南非洲地区的人类昏睡病和牛的纳加纳病。当在哺乳动物宿主中生长时，布氏锥虫细胞会定期转换其主要表面糖蛋白，以逃避宿主免疫系统，这种现象称为抗原变异。这些表面糖蛋白仅从位于染色体末端附近的约 20 个基因座之一（端粒）表达。酵母中的端粒形成一种特殊的结构，影响附近基因的转录。在布氏锥虫中也观察到类似的现象，端粒可能在抗原变异中发挥重要作用。布氏锥虫端粒研究的最终目标是了解抗原变异中的端粒功能，这是布氏锥虫发病机制的一个重要方面。基本步骤是识别端粒成分并表征其功能，这是该提案的主要目标。具体目标 1：使用两种方法纯化 tbTRF（布氏锥虫中的双链端粒 DMA 结合因子）蛋白复合物：1) 使用抗 FLAG 和抗 HA 单克隆抗体对 FLAG-HA-HA 标记的 tbTRF 进行顺序免疫沉淀下拉。免疫共沉淀的蛋白质将通过质谱法进行鉴定。 2) 使用 lexA-tbTRF 作为诱饵和 T. brucei GAD 融合 cDNA 文库进行酵母 2-杂交筛选。 tbTRF 相互作用因子的候选者将首先通过免疫共沉淀来确认它们在体内与 tbTRF 的相互作用。通过检查敲除或 RNAi 敲除细胞系中的表型，将表征阳性克隆在抗原变异和端粒功能中的作用。具体目标 2：选择 tbTRFH 结构域中非致命相互作用缺陷的 tbTRF 突变，以更好地表征 tbTRF 的功能。使用 PCR 克隆，将在 tbTRFH 结构域中产生随机点突变或小缺失。失去与野生型 tbTRF 相互作用但仍保留与其相互作用的能力的突变体将使用常规和反向 2-杂交分析进行筛选。敲入或过度表达这些突变等位基因的细胞系将以其抗原变异的表型为特征。端粒复合体成分的鉴定及其功能的后续表征将是研究抗原变异中端粒功能的良好起点，抗原变异是布氏锥虫病原体的一个重要方面。