Multifunctional class I transcription in T. brucei

描述(申请人提供)：布氏锥虫原生寄生虫通过采采蝇媒介传播，在血液中自由生活，在人类中引起致命的非洲睡眠病，在各种牲畜中引起类似的疾病Nagana。治疗这种疾病的药物只有四种，其中最有效的药物美拉索洛尔毒性很高，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。寄生虫与宿主的界面是细胞表面一层致密的糖蛋白涂层，它由寄生在哺乳动物血液中的单一类型的变异表面糖蛋白(VSG)和在苍蝇中肠中繁殖的原环素组成。被毛保护寄生虫免受裂解宿主成分的伤害，VSG被膜的抗原变异是寄生虫逃避免疫反应的手段。从单个VSG基因以极高的效率表达了完整的VSG外壳。这一表达水平对寄生虫至关重要，因为沉默VSG的表达会导致寄生虫在培养中迅速停止生长，并有效地清除受感染的小鼠的锥虫。布鲁氏毛滴虫已经进化出一种独特的多功能RNA聚合酶(PolI)系统，可以有效地转录VSG和ProCyclin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上，参与依赖于发育的转录调控，并隔离在两个不同的亚核区。因此，这种前所未有的多功能性很可能需要寄生虫所特有的基本蛋白质、蛋白质结构域和蛋白质-蛋白质相互作用，而这些是寄生虫所没有的。作为寄生虫特有的特征，到目前为止，我们已经鉴定了新的多亚基转录因子CITFA，它是I类转录和RNA PolI所必需的，RPA31是RPA2亚基中不寻常的N末端延伸域，以及RPB5、RPB6和RPB10共同亚基的变体集，RPB5、RPB6和RPB10是RNA PolI特有的。作为小分子抑制研究的先决条件，我们将继续从功能上表征这些独特的蛋白质，并分析它们的蛋白质-蛋白质相互作用。此外，我们将利用我们最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：这一应用的拟议研究将探索对致命性寄生虫布氏锥虫表达其主要细胞表面抗原并在其人类和媒介宿主中生存不可或缺的转录因子。因此，这些因素对寄生虫的生长至关重要。由于其中一些似乎是针对这种寄生虫的，它们是潜在的化疗新靶点。迫切需要这样的靶点，因为治愈布氏毛滴虫感染的药物很少，而且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.