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（由申请方提供）：布氏锥虫是一种原生动物寄生虫，可引起人类非洲锥虫病和牛长角线虫病。在哺乳动物宿主中，血流形式（BF）T。布氏杆菌经历抗原变异，并有规律地转换其表面抗原，即变异表面糖蛋白（VSG），以逃避宿主的免疫攻击。虽然T.布氏杆菌具有上千个VSG基因和假基因，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转换的有效性并使其效率最大化。因此，VSG转换和单等位基因VSG表达对于T.布氏菌致病性端粒与VSG的表达位点相邻，长期以来一直被认为在VSG的表达调控中起重要作用。事实上，已经表明，在酵母，人类和T。在布氏细胞中，端粒形成异染色质结构，其影响靶向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RAP 1。为了探讨端粒在VSG表达和转换调控中的作用，我们一直致力于T。布氏杆菌端粒特异性蛋白及其功能的鉴定。我们克隆了第一个T.布氏杆菌端粒结合蛋白。更重要的是，我们最近发现了T。利用tbTRF作为诱饵，在酵母双杂交筛选中鉴定了布氏杆菌RAP 1，证实了tbRAP 1是端粒复合物的内在组分，并证明了它对于沉默BF T中的亚端粒VSG是必需的。布氏细胞这一发现揭示了T.布鲁氏菌端粒作为表面抗原表达控制的关键参与者，并将tbRAP 1鉴定为抗寄生虫药物的潜在靶标。我们的发现也表明T.布氏杆菌与包括恶性疟原虫和C. glabrata中，端粒沉默在毒力基因表达调控中起重要作用。我们计划进一步研究端粒蛋白，特别是tbRAP 1和tbTRF，如何调节VSG的表达和转换。本研究为最终根除T.布氏杆菌和其他类似的微生物病原体。我们提出了几种方法来进一步研究tbRAP 1和tbTRF的功能。首先，我们的观察表明，定位tbRAP 1的端粒是至关重要的VSG沉默功能。因此，我们假设，如果tbRAP 1直接结合端粒DNA，这种活动将是至关重要的VSG沉默。然而，如果tbRAP 1缺乏任何DNA结合活性，则tbRAP 1和tbTRF之间的相互作用以及tbTRF的端粒结合功能对于将tbRAP 1锚定到端粒是必需的。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tbRAP 1介导的沉默的关键机制，并阐明tbRAP 1和其他RAP 1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的功能。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tbRAP 1的功能。首先，我们假设tbRAP 1不仅参与VSG沉默控制，还影响VSG切换率，这将在目标2.1中进行测试。其次，我们将阐明tbRAP 1的不同功能之间的关系，并确定哪些领域的tbRAP 1是必不可少的VSG表达和/或开关调节使用系统的遗传方法在目标2.2。这些研究将有助于揭示tbRAP 1在抗原变异中的作用机制。第三，我们假设tbRAP 1通过调节染色质结构来应用其沉默效应。因此，我们将研究是否tbRAP 1优先与沉默的染色质和是否tbRAP 1的损失会导致任何变化的染色质结构的去阻遏的ES。最后，我们假设tbRAP 1工程与其他未知的辅助因子建立/维持沉默结构在subtelomeric位点。为了寻找tbRAP 1相互作用的因素，也发挥重要作用的抗原变异，我们的目标是确定tbRAP 1蛋白复合物的组成部分，通过顺序免疫沉淀。研究tbRAP 1介导的沉默通路中其他因子的功能将有助于我们更好地理解其潜在机制。在tbRAP 1相互作用的候选人中，我们可以确定参与VSG沉默/开关的下游效应子和参与tbRAP 1的表达、稳定性或活性调节的蛋白质。识别参与抗原变异的新因子也为抗寄生虫药物提供了更多潜在的靶点，并有助于最终消除这种寄生虫。