Impact of telomere-associated protein complexes on VSG expression site regulation and structure in Trypanosoma brucei

原生病原体布鲁氏锥虫是撒哈拉以南非洲地区人类昏睡病和牲畜纳迦纳病等致命疾病的病原体。在它们的生命周期中，锥虫必须在哺乳动物宿主和采采蝇载体之间穿梭。在哺乳动物宿主中，布氏体血状细胞（BSF）的免疫逃避依赖于抗原变异，包括单等位基因表达和变异表面糖蛋白（VSG）基因的周期性开关。活性VSG总是从15个亚端粒表达位点（ESs）中的一个转录，并且端粒相关蛋白已被证明调节这些位点的表达和重组事件。在从BSF向昆虫常驻原环型（PCF）分化过程中，活性ES在转录上被沉默。调控抗原变异的确切分子机制仍然难以捉摸，因为迄今为止只有少数端粒相关蛋白被识别和表征。我们使用定量相互作用组学方法确定了布鲁氏体端粒蛋白复合物的组成，并鉴定了17种端粒相关蛋白，其中包括已经验证的端粒蛋白。在这项拨款申请中，我们提出了两个独立但互补的项目，以表征新的端粒相关蛋白，并阐明它们在抗原性变异和端粒维持中的假定功能。初步数据已经表明，在两种形式的寄生虫中存在不同的端粒复合物组成。为了验证我们的定量相互作用学方法，我们已经证明了一种新的端粒结合蛋白TelAP1与端粒蛋白TRF、RAP1和TIF2形成复合物，并在发育分化过程中影响ES沉默动力学。

The protist pathogen, Trypanosoma brucei, is the causative agent of fatal diseases such as sleeping sickness in humans and nagana in livestock in sub-Saharan Africa. During their life cycle, trypanosomes have to shuttle between a mammalian host and the tsetse fly vector. In the mammalian host, immune evasion of T. brucei bloodstream form cells (BSF) relies on antigenic variation, which includes monoallelic expression and periodic switching of variant surface glycoprotein (VSG) genes. The active VSG is always transcribed from one of only 15 subtelomeric expression sites (ESs) and telomere-associated proteins have been shown to regulate expression and recombination events at these sites. During differentiation from BSF to the insect-resident procyclic form (PCF), the active ES is transcriptionally silenced. The exact molecular mechanisms that regulate antigenic variation are still elusive because only a few telomere-associated proteins have been identified and characterized so far.We used quantitative interactomics to determine the composition of telomere protein complexes in T. brucei and identified 17 telomere-associated proteins including already validated telomeric proteins. In this grant proposal, we suggest two independent but complementary projects to characterise novel telomere-associated proteins and elucidate their putative function in antigenic variation and telomere maintenance. Preliminary data already suggest different telomere complex compositions in the two forms of the parasite. To validate our quantitative interactomics approach, we could already show that one of the novel telomere-binding proteins, TelAP1, forms a complex with telomeric proteins TRF, RAP1 and TIF2 and influences ES silencing kinetics during developmental differentiation.