Multifunctional class I transcription in T. brucei

描述（由申请人提供）：原生生物寄生虫布氏锥虫通过采采蝇媒介传播，在血液中自由生活，导致人类致命疾病非洲昏睡病和各种牲畜的类似疾病Nagana。治疗这种疾病的药物只有四种，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变得越来越重要。寄生虫与其宿主的界面是细胞表面上的致密糖蛋白涂层，其由寄生于哺乳动物血流的形式的单一类型的变体表面糖蛋白（VSG）和在苍蝇中肠中增殖的形式的原环素组成。该外壳保护寄生虫免受裂解宿主组分的侵害，并且VSG外壳的抗原变异是寄生虫逃避免疫应答的手段。完整的VSG外壳由单个VSG基因以极高的效率表达。这种表达水平对于寄生虫是至关重要的，因为沉默VSG表达导致培养物中寄生虫生长的快速停止和从感染小鼠中有效清除锥虫。T.布氏杆菌已经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大的核糖体基因单位，而在T。在布鲁氏菌中，RNA pol I被募集到四个结构不同的启动子，参与发育依赖性转录调控，并被隔离到两个不同的亚核区室中。因此，这是最有可能的，这种前所未有的多功能性需要必需的蛋白质，蛋白质结构域和蛋白质-蛋白质相互作用，是独特的寄生虫和主机中不存在。作为寄生虫特异性特征，我们迄今为止已经表征了对于I类转录和RNA pol I不可或缺的新型多亚基转录因子CITFA、新型和必需亚基RPA 31、亚基RPA 2中不寻常的N-末端延伸结构域以及对RNA pol I特异的常见亚基RPB 5、RPB 6和RPB 10的变体集。作为小分子抑制研究的先决条件，我们将继续对这些独特的蛋白质进行功能表征，并分析它们的蛋白质-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来鉴定新的I类转录因子。公共卫生相关性：本申请的拟议研究将探索致命寄生虫布氏锥虫表达其主要细胞表面抗原并在其人类和载体宿主中生存所不可或缺的转录因子。因此，这些因素对寄生虫生长至关重要。由于它们中的一些似乎对寄生虫具有特异性，因此它们是化疗的潜在新靶点。这样的靶点是治疗结核病的急需药物.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。

DESCRIPTION (provided by applicant): 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 RELEVANCE: 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.