Multifunctional class I transcription in T. brucei

摘要 布氏锥虫原生寄生虫通过采采蝇媒介传播，自由生活在 血液流动，并导致致命的疾病非洲睡眠病和类似的疾病 在各种家畜中都有Nagana。治疗这种疾病的药物只有四种，而最有效的药物， 美拉索洛尔是一种剧毒药物，寄生虫对这种药物的耐药性正在上升。因此，它就成了 寻找新的抗寄生虫靶点和开发新的治疗策略日益重要。这个 寄生虫与宿主的界面是细胞表面的一层致密的糖蛋白涂层，它由一个单一的 一种变异型表面糖蛋白(VSG)，其形式寄生于哺乳动物的血流和 原环素的形式在苍蝇中肠中繁殖。毛皮保护寄生虫免受裂解宿主的侵袭。 VSG外壳的成分和抗原变异是寄生虫逃避免疫的手段 回应。从单个VSG基因以极高的效率表达了完整的VSG外壳。这 表达水平对寄生虫至关重要，因为沉默VSG表达会导致迅速停止 寄生虫在培养中的生长和从感染的小鼠中有效清除锥虫。布氏毛滴虫 进化出一种独特的多功能RNA聚合酶(PolI)系统，有效地转录VSG和 原周期蛋白基因(I类转录)。在其他真核生物中，这种有效的酶只转录 大的核糖体基因单位而在布鲁氏锥虫中，rna polI被招募到四个结构不同的 启动子，参与依赖发育的转录调控，并隔离为两个 不同的亚核区。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 在东道主中缺席。作为寄生虫特有的特征，到目前为止，我们已经将这种新颖的多亚基描述为 转录因子CITFA是I类转录和RNA PolI所必需的，新的和 基本亚基RPA31，亚基RPA2中的一个不寻常的N末端延伸域，以及 针对RNA polI的共同亚基RPB5、RPB6和RPB10。作为小分子 分子抑制研究，我们将继续对这些独特的蛋白质和 分析它们的蛋白质-蛋白质相互作用。此外，我们还将开发我们新开发的蛋白质 纯化技术鉴定新的I类转录因子。《公共卫生声明》 对这一应用的拟议研究将探索转录因子，这些转录因子是 致死寄生虫布氏锥虫表达其主要细胞表面抗原并在人和 向量宿主。因此，这些因素对寄生虫的生长至关重要。因为其中一些似乎是 针对这种寄生虫，它们是潜在的化疗新靶点。这样的目标是迫切需要的- 导致治愈布氏毛滴虫感染的药物很少，对这些药物的毒性和寄生虫耐药性正在上升。

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.