Multifunctional class I transcription in T. brucei

总结 原生生物寄生虫布氏锥虫由采采蝇传播，自由生活在 血液和导致致命的疾病非洲睡眠病在人类和类似的疾病 在各种牲畜中的Nagana。只有四种药物治疗这种疾病，最有效的药物， 美拉胂醇毒性很强，寄生虫对这种药物的抗药性正在上升。因此， 寻找新的抗寄生虫靶点和开发新的治疗策略越来越重要。的 寄生虫与其宿主的界面是细胞表面上的致密糖蛋白涂层， 以寄生于哺乳动物血流的形式存在的变异表面糖蛋白（VSG）类型， 原环素在果蝇中肠中繁殖。外套保护寄生虫免受溶菌宿主的侵害 成分和抗原变异是寄生虫逃避免疫的手段 反应完整的VSG外壳由单个VSG基因以极高的效率表达。这 表达水平对寄生虫是至关重要的，因为沉默VSG表达会导致寄生虫的快速停止。 寄生虫在培养物中的生长和从感染的小鼠中有效清除锥虫。t.布鲁塞 进化出一种独特的多功能RNA聚合酶（pol）I系统，以有效地转录VSG和 原细胞周期蛋白基因（I类转录）。在其他真核生物中，这种高效的酶只转录 核糖体基因大单位，而在T.在布氏杆菌中，RNA聚合酶I被募集到四种结构不同的 启动子，参与发育依赖性转录调控，并被隔离成两个 不同的亚核区室。因此，这种前所未有的多功能性很可能需要 寄生虫特有的必需蛋白质、蛋白质结构域和蛋白质-蛋白质相互作用， 缺席的主持人。作为寄生虫特异性特征，我们迄今为止已经表征了新的多亚基， CITFA是I类转录和RNA聚合酶I所必需的转录因子， 必需亚基RPA31，亚基RPA2中不常见的N-末端延伸结构域，以及一组变异的 对RNA pol I具有特异性的共同亚基RPB 5、RPB 6和RPB 10。作为小的先决条件， 分子抑制研究，我们将继续功能特性这些独特的蛋白质， 分析它们的蛋白质相互作用。此外，我们将利用我们新开发的蛋白质 纯化技术以鉴定新的I类转录因子。公共卫生声明 这项应用的拟议研究将探索转录因子，这些转录因子对于转录过程中不可或缺。 致死性寄生虫布氏锥虫表达其主要细胞表面抗原并在其人体内存活， 病媒宿主。因此，这些因素对寄生虫生长至关重要。因为其中一些似乎是 它们是寄生虫特有的，是潜在的化疗新靶点。这些目标迫切需要- 导致药物治愈T布氏杆菌感染很少，对这些药物的毒性和寄生虫抗药性正在上升。

SUMMARY The protistan parasite Trypanosoma brucei is transmitted by the tsetse vector, lives freely in the bloodstream and causes the lethal disease African Sleeping Sickness in humans and the similar disease Nagana in various livestock. There are only four drugs for this disease, and the most effective drug, melarsoprol, is highly toxic and parasite resistance to this drug is on the rise. Thus, it becomes increasingly important to find new anti-parasitic targets and develop new therapeutic strategies. The parasite's interface to its hosts is a dense glycoprotein coat on the cell surface which consists of a single type of variant surface glycoprotein (VSG) in the form parasitizing the mammalian bloodstream and of procyclin in the form multiplying in the fly midgut. The coat protects the parasite from lytic host components and antigenic variation of the VSG coat is the parasite's means to evade the immune response. The complete VSG coat is expressed with extreme efficiency from a single VSG gene. This expression level is crucial for the parasite because silencing VSG expression leads to rapid cessation of parasite growth in culture and effective clearance of trypanosomes from infected mice. T. brucei has evolved a unique multifunctional RNA polymerase (pol) I system to effectively transcribe both VSG and Procyclin genes (class I transcription). In other eukaryotes, this efficient enzyme transcribes exclusively the large ribosomal gene unit whereas in T. brucei, RNA pol I is recruited to four structurally different promoters, involved in development-dependent transcriptional regulation, and sequestered into two distinct subnuclear compartments. Hence, it is most likely that this unprecedented versatility requires essential proteins, protein domains and protein-protein interactions that are unique to the parasite and absent in the hosts. As parasite-specific features, we have thus far characterized the novel, multi-subunit transcription factor CITFA that is indispensable for class I transcription and for RNA pol I, the novel and essential subunit RPA31, an unusual N-terminal extension domain in subunit RPA2, and a variant set of the common subunits RPB5, RPB6 and RPB10 that is specific to RNA pol I. As a prerequisite for small molecule inhibition studies, we will proceed to functionally characterize these unique proteins and analyze their protein-protein interactions. In addition, we will exploit our newly developed protein purification technology to identify new class I transcription factors. Public Health Statement The proposed studies of this application will explore transcription factors that are indispensable for the lethal parasite Trypanosoma brucei to express its major cell surface antigens and to survive in its human and vector hosts. Accordingly, these factors are essential for parasite growth. Since some of them appear to be specific to the parasite, they are potential new targets for chemotherapy. Such targets are urgently needed be- cause drugs to cure a T. brucei infection are few and toxic and parasite resistance to these drugs is on the rise.