Mitochondrial poly(A) Polymerases of Trypanosomes

描述(申请人提供)：锥虫是一种原生动物血鞭毛虫，可导致多种人类疾病，包括恰加斯病、利什曼病和非洲昏睡病，这些疾病每年造成近100万人死亡。寄生虫巨大线粒体中独特的遗传现象已被广泛研究，在理解运动基质DNA(KDNA)复制和尿苷插入/缺失mRNA编辑方面取得了重大进展。然而，控制线粒体中信使核糖核酸稳定性和周转的分子机制大多仍不清楚。我们的目标是为控制线粒体mRNA聚腺苷酸化的调控机制的分子分析奠定基础，并更好地了解kDNA表达与生命周期相关的变化。支持这项研究的特定假设是，锥虫线粒体中的多聚腺苷酸化对具有翻译能力的mRNAs的稳定性至关重要，因此，线粒体的功能也是如此。我们认为，多聚腺苷的形成是通过作为多蛋白复合体催化亚单位的动泡体多(A)聚合酶(KPAP1)完成的。KPAP1已经在这一提议的初步实验过程中通过与RNA尿苷酰转移酶(TUTase)的同源性而被鉴定。我们的发现将TUTase样结构支架的已知作用扩展到参与线粒体RNA加工的ATP特异性酶。我们建议：1.表征kPAP1在未编辑、编辑前和编辑后的mRNA多腺化中的作用。RNA干扰将被用来下调原循环和血流形式的kPAPs的表达，它们对线粒体功能的意义将通过对细胞活力、膜电位和RNA转录本的比较分析来解决。2.阐明锥虫线粒体多聚腺苷酸化机制的蛋白质组成。含有kPAP1的复合体将通过串联亲和层析分离、酶活性测试和质谱分析。 简介：锥虫是世界各地发展中国家主要寄生虫病的病原体。考虑到驻扎在波斯湾和阿富汗的美军中内脏利什曼病的发生，美国对有效的抗锥虫疗法的需求也是可以预见的。现有的治疗方法往往是有毒和无效的，这就产生了对新药发现的需求。靶向基本的寄生虫特异性酶，如我们的提案中首次描述的线粒体聚(A)聚合酶，是创造新一代抗锥虫药物的一种有前途的方法。然而，需要对聚腺苷酸化过程进行详细的分子分析，以验证该酶作为药物靶标的有效性。

DESCRIPTION (provided by applicant): Trypanosomatids are protozoan hemoflagellates responsible for a variety of human diseases, including Chagas' disease, leishmaniasis, and African sleeping sickness, which account for nearly one million deaths per year. The unique genetic phenomena occurring in parasites' giant mitochondria have been extensively studied, producing major advances in the understanding of kinetoplast DNA (kDNA) replication and uridine insertion/deletion mRNA editing. However, the molecular mechanisms governing mRNA stability and turnover in mitochondria remain mostly unknown. Our goal is to set the stage for molecular analysis of the regulatory mechanisms controlling mitochondrial mRNA polyadenylation, and better understanding of life cycle- correlated changes in kDNA expression. The specific hypothesis underlying the proposed research is that polyadenylation in trypanosomal mitochondria is essential for the stability of translationally-competent mRNAs and, therefore, mitochondrial function. We suggest that polyadenylation is accomplished by a Kinetoplast Poly(A) Polymerase (kPAP1) acting as catalytic subunit of multi-protein complex. KPAP1 has been identified in the course of preliminary experiments for this proposal via homology with RNA uridylyl transferases (TUTases). Our findings expand known roles of TUTase-like structural scaffolds to ATP-specific enzymes involved in mitochondrial RNA processing. We propose to: 1. Characterize the roles of kPAP1 in polyadenylation of non-edited, pre-edited, and edited mRNAs. RNA interference will be used to down-regulate expression of both kPAPs in procyclic and bloodstream forms, and their significance for mitochondrial function will be addressed by comparative analysis of cell viability, membrane potential, and RNA transcripts. 2. Elucidate the protein composition of the polyadenylation machinery in trypanosomal mitochondria. The kPAP1-containing complexes will be isolated by tandem affinity chromatography, tested for enzymatic activity, and analyzed by mass spectrometry. Narrative: Trypanosomatids are the causative agents of major parasitic diseases in developing countries around the world. Considering the occurrence of visceral leishmaniasis among U.S. troops stationed in the Persian Gulf and Afghanistan, a demand for effective anti- trypanosomal therapies also may be anticipated in the United States. Available treatments are often toxic and ineffective, creating demand for the discovery of new drugs. Targeting essential parasite-specific enzymes, such as the mitochondrial poly(A) polymerase described for the first time in our proposal, is a promising approach toward creating a new generation of trypanocides. However, a detailed molecular analysis of the polyadenylation process is required for validation of this enzyme as a drug target.