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）：Brucei锥虫是一种原生动物寄生虫，可引起人类和牛长老的非洲锥虫病。在哺乳动物宿主中，血液形式（BF）T。Brucei经历抗原变异，并定期切换其表面抗原变异表面糖蛋白（VSG），以逃避宿主的免疫攻击。尽管布鲁氏菌具有超过一千个VSG基因和假基因，但VSG以严格的单相关方式仅从亚端粒基因座表达，这确保了VSG切换的有效性并最大程度地提高其效率。因此，VSG开关和单位型VSG表达对于T. brucei发病机理至关重要。端粒与VSG的表达位点相邻，长期以来一直提出在VSG表达调节中起重要作用。实际上，已经表明，在酵母，人和田氏菌细胞中，端粒形成了一种异性结构，该结构影响了针对亚端子群的报告基因的转录。特别是在酵母中，这种端粒沉默已被证明取决于端粒蛋白RAP1。为了探索VSG表达和切换调节中的端粒功能，我们一直专注于识别Brucei端粒特异性蛋白质及其功能的表征。我们克隆了第一个t. brucei端粒结合蛋白TBTRF。更重要的是，我们最近使用TBTRF作为诱饵在酵母2杂交筛网中鉴定出T. brucei Rap1，证实TBRAP1是端粒复合物的固有成分，证明它对于在BF T. Brucei细胞中的静音亚telomeric vsgs至关重要。这一发现揭示了布鲁氏菌端粒是表面抗原表达控制的关键参与者，并将TBRAP1鉴定为抗寄生虫药物的潜在靶标。我们的发现还表明，布鲁氏菌类似于其他几种病原体，包括恶性疟原虫和glabrata，其中端粒沉默在调节毒力基因表达中起着重要作用。我们计划进一步研究端粒蛋白（特定TBRAP1和TBTRF）如何调节VSG表达和切换。这项研究将有助于最终消除布鲁氏菌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，我们的观察结果表明，将TBRAP1定位到端粒对其VSG沉默函数至关重要。因此，我们假设TBRAP1直接结合端粒DNA，则该活性对于VSG沉默至关重要。但是，如果TBRAP1缺乏任何DNA结合活性，则TBRAP1和TBTRF与TBTRF的端粒结合函数之间的相互作用对于将TBRAP1锚定在端粒上至关重要。我们将使用体外方法在特定目标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TNA结合和蛋白质 - 蛋白质相互作用的相似和独特特征的TBRAP1和其他RAP1同源物。因此，我们的工作将有助于确定抗寄生虫药物的潜在靶标。在特定目标2中，我们旨在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不仅参与了VSG误解控制，而且还会影响VSG开关率，这将在AIM 2.1中进行测试。其次，我们将阐明TBRAP1的不同功能之间的关系，并确定TBRAP1的哪些域对于使用AIM 2.2中的系统遗传方法进行VSG表达和/或切换调节至关重要。这些研究将有助于揭示TBRAP1在抗原变异中功能的潜在机制。第三，我们假设TBRAP1通过调节染色质结构来应用其沉默效果。因此，我们将检查TBRAP1是否优先将其与无声染色质相关联，以及TBRAP1的丢失是否会导致过压缩ESS的染色质结构的任何变化。最后，我们假设TBRAP1与其他未知的共同因素一起工作，以建立/维持亚电位基因座的沉默结构。为了搜索在抗原变异中也起着重要作用的TBRAP1相互作用因子，我们旨在通过顺序免疫沉淀来鉴定TBRAP1蛋白复合物的成分。在同一TBRAP1介导的沉默途径中研究其他因素的功能将有助于我们更好地了解潜在的机制。在TBRAP1相互作用的候选者中，我们可以鉴定参与VSG沉默/开关和蛋白质的下游效应子，以及涉及TBRAP1表达，稳定性或活性的蛋白质。鉴定抗原变异的新因素还为抗寄生虫药物提供了更多的潜在靶标，并有助于最终消除该寄生虫。