Characterize functions of T. brucei RAP1 and TRF in antigenic variation and telom

表征 T. brucei RAP1 和 TRF 在抗原变异和端粒中的功能

• 批准号: 8603220
• 负责人: Bibo Li
• 金额: $ 35.5万
• 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 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8603220
• 关键词: Affect; Africa; African; African Trypanosomiasis; Antigenic Variation; Binding; Blood Circulation; Candida glabrata; Cattle; Cells; Chagas Disease; Chromatin; Chromatin Structure; Chromosomes; Complex; Country; DNA; DNA Binding; DNA-Binding Proteins; Disease; Distal; Economic Development; Effectiveness; Ensure; Future; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genitourinary system; Health; Homologous Gene; Human; Hybrids; Immune; Immunoprecipitation; In Vitro; Incidence; Infection; Link; Livestock; Maintenance; Malaria; Mediating; Membrane Glycoproteins; Nucleoproteins; Parasites; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Plasmodium falciparum; Play; Proteins; Pseudogenes; Recruitment Activity; Regulation; Reporter Genes; Role; Sahara; Sepsis; Series; Site; Structure; Surface Antigens; Telomere-Binding Proteins; Testing; Time; Trypanosoma brucei brucei; Trypanosoma cruzi; Variant; Virulence; Work; Yeasts; genetic analysis; in vivo; microbial; mortality; mutant; nagana; novel; pathogen; protein complex; protein protein interaction; rural area; telomere

DESCRIPTION (provided by applicant): Trypanosoma brucei is a protozoan parasite that causes African trypanosomiasis in humans and nagana in cattle. In mammalian hosts, bloodstream form (BF) T. brucei undergoes antigenic variation and regularly switches its surface antigen, variant surface glycoprotein (VSG), to evade the host's immune attack. Although T. brucei has more than a thousand VSG genes and pseudogenes, VSG is expressed exclusively from subtelomeric loci in a strictly monoallelic manner, which ensures effectiveness of VSG switching and maximizes its efficiency. Therefore, VSG switching and monoallelic VSG expression are essential for T. brucei pathogenesis. Telomeres, being adjacent to the expression sites of VSGs, have long been proposed to play an important role in VSG expression regulation. Indeed, it has been shown that in yeast, human, and T. brucei cells, telomeres form a heterochromatic structure that affects the transcription of reporter genes targeted to subtelomeres. Particularly in yeast, this telomeric silencing has been shown to depend on telomere protein RAP1. To explore telomere functions in VSG expression and switching regulation, we have been focusing on identification of T. brucei telomere-specific proteins and characterization of their functions. We have cloned the first T. brucei telomere-binding protein, tbTRF. More importantly, we have recently identified T. brucei RAP1 in a yeast 2-hybrid screen using tbTRF as bait, confirmed that tbRAP1 is an intrinsic component of the telomere complex, and demonstrated that it is essential for silencing subtelomeric VSGs in BF T. brucei cells. This discovery reveals T. brucei telomere as a key player in surface antigen expression control and identifies tbRAP1 as a potential target for anti-parasite drugs. Our finding also shows that T. brucei is similar to a couple of other pathogens including P. falciparum and C. glabrata, in which telomeric silencing plays an important role in regulation of virulence gene expression. We plan to further study how telomere proteins, particular tbRAP1 and tbTRF, regulate VSG expression and switching. This study would be helpful for eventual eradication of T. brucei and other similar microbial pathogens. We propose several approaches to further study the functions of tbRAP1 and tbTRF. First, our observations suggest that localization of tbRAP1 to the telomere is crucial for its VSG silencing function. We therefore hypothesize that if tbRAP1 binds telomere DNA directly, this activity would be critical for VSG silencing. However, if tbRAP1 lacks any DNA binding activity, the interaction between tbRAP1 and tbTRF and the telomere binding function of tbTRF would be essential for anchoring tbRAP1 to the telomere. We will test these hypotheses in Specific Aim 1 using in vitro approaches. These studies will reveal a key mechanism for tbRAP1-mediated silencing and elucidate similar and unique features of the DNA binding and protein-protein interaction functions of tbRAP1 and other RAP1 homologs. Our work would therefore help to identify potential targets for anti-parasite drugs. In Specific Aim 2, we aim to carry out a series of in vivo genetic analyses to further understand tbRAP1's function. First, we hypothesize that tbRAP1 not only participates in VSG-silencing control but also influences VSG switching rates, which will be tested in Aim 2.1. Second, we will elucidate the relationship between different functions of tbRAP1 and to determine which domains of tbRAP1 are essential for VSG expression and/or switching regulation using systematic genetic approaches in Aim 2.2. These studies will help to reveal the underlying mechanisms of tbRAP1's function in antigenic variation. Third, we hypothesize that tbRAP1 applies its silencing effect by modulating the chromatin structure. We will therefore examine whether tbRAP1 preferentially associates with the silent chromatin and whether loss of tbRAP1 causes any changes in the chromatin structure of the derepressed ESs. Last, we hypothesize that tbRAP1 works with other unknown co-factors to establish/maintain the silencing structure at subtelomeric loci. In order to search for tbRAP1-interacting factors that also play important roles in antigenic variation, we aim to identify components of tbRAP1 protein complex by sequential immunoprecipitation. Studying functions of other factors in the same tbRAP1-mediated silencing pathway will help us to better understand the underlying mechanisms. Among the tbRAP1-interacting candidates, we may identify downstream effectors involved in VSG silencing/switching and proteins involved in the regulation of the expression, stability, or activity of tbRAP1. Identification of novel factors involved in antigenic variation also provides more potential targets for anti-parasite drugs and helps for eventual elimination of this parasite.

描述（由申请人提供）：布氏锥虫是一种原生动物寄生虫，可引起人类的非洲锥虫病和牛的那加那虫病。在哺乳动物宿主中，血流形式（BF）布氏锥虫经历抗原变异并定期转换其表面抗原，即变体表面糖蛋白（VSG），以逃避宿主的免疫攻击。尽管布氏锥虫有一千多个VSG基因和假基因，但VSG仅以严格的单等位基因方式从亚端粒位点表达，这确保了VSG转换的有效性并最大化其效率。因此，VSG 转换和单等位基因 VSG 表达对于布氏锥虫的发病机制至关重要。端粒邻近 VSG 的表达位点，长期以来一直被认为在 VSG 表达调节中发挥重要作用。事实上，已经表明，在酵母、人类和布氏锥虫细胞中，端粒形成异染色质结构，影响针对亚端粒的报告基因的转录。特别是在酵母中，这种端粒沉默已被证明依赖于端粒蛋白 RAP1。为了探索 VSG 表达和转换调节中的端粒功能，我们一直专注于 T. brucei 端粒特异性蛋白的鉴定及其功能表征。我们克隆了第一个布氏锥虫端粒结合蛋白 tbTRF。更重要的是，我们最近在使用 tbTRF 作为诱饵的酵母 2-hybrid 筛选中鉴定了 T. brucei RAP1，证实 tbRAP1 是端粒复合物的内在成分，并证明它对于沉默 BF T. brucei 细胞中的亚端粒 VSG 至关重要。这一发现揭示了布氏锥虫端粒在表面抗原表达控制中的关键作用，并将 tbRAP1 确定为抗寄生虫药物的潜在靶点。我们的发现还表明，布氏锥虫与恶性疟原虫和光滑念珠菌等几种其他病原体相似，其中端粒沉默在毒力基因表达的调节中发挥着重要作用。我们计划进一步研究端粒蛋白，特别是 tbRAP1 和 tbTRF 如何调节 VSG 表达和转换。这项研究将有助于最终根除布氏锥虫和其他类似的微生物病原体。我们提出了几种进一步研究 tbRAP1 和 tbTRF 功能的方法。首先，我们的观察表明 tbRAP1 端粒的定位对于其 VSG 沉默功能至关重要。因此，我们假设如果 tbRAP1 直接结合端粒 DNA，这种活性对于 VSG 沉默至关重要。然而，如果 tbRAP1 缺乏任何 DNA 结合活性，则 tbRAP1 和 tbTRF 之间的相互作用以及 tbTRF 的端粒结合功能对于将 tbRAP1 锚定到端粒至关重要。我们将使用体外方法在具体目标 1 中测试这些假设。这些研究将揭示 tbRAP1 介导的沉默的关键机制，并阐明 tbRAP1 和其他 RAP1 同源物的 DNA 结合和蛋白质-蛋白质相互作用功能的相似和独特特征。因此，我们的工作将有助于确定抗寄生虫药物的潜在靶点。在具体目标2中，我们的目标是进行一系列体内遗传分析，以进一步了解tbRAP1的功能。首先，我们假设tbRAP1不仅参与VSG沉默控制，而且影响VSG切换率，这将在目标2.1中进行测试。其次，我们将阐明 tbRAP1 不同功能之间的关系，并使用目标 2.2 中的系统遗传学方法确定 tbRAP1 的哪些结构域对于 VSG 表达和/或转换调节至关重要。这些研究将有助于揭示 tbRAP1 在抗原变异中发挥作用的潜在机制。第三，我们假设 tbRAP1 通过调节染色质结构来发挥其沉默作用。因此，我们将检查 tbRAP1 是否优先与沉默染色质结合，以及 tbRAP1 的丢失是否会导致去抑制 ES 的染色质结构发生任何变化。最后，我们假设 tbRAP1 与其他未知辅助因子一起建立/维持亚端粒位点的沉默结构。为了寻找在抗原变异中也发挥重要作用的 tbRAP1 相互作用因子，我们的目标是通过序贯免疫沉淀来鉴定 tbRAP1 蛋白复合物的成分。研究同一 tbRAP1 介导的沉默途径中其他因素的功能将有助于我们更好地理解潜在的机制。在 tbRAP1 相互作用的候选者中，我们可以鉴定参与 VSG 沉默/转换的下游效应子以及参与 tbRAP1 表达、稳定性或活性调节的蛋白质。识别与抗原变异相关的新因子也为抗寄生虫药物提供了更多潜在靶点，并有助于最终消除这种寄生虫。