Mitochondrial poly(A) Polymerases of Trypanosomes

描述（由申请人提供）：锥虫是一种原生动物血鞭毛虫，可引起多种人类疾病，包括恰加斯病、利什曼病和非洲昏睡病，每年造成近100万人死亡。寄生虫巨大线粒体中发生的独特遗传现象已被广泛研究，在对动质体DNA（kDNA）复制和尿苷插入/缺失mRNA编辑的理解方面取得了重大进展。然而，线粒体中mRNA稳定性和周转的分子机制仍然是未知的。我们的目标是为控制线粒体mRNA多聚腺苷酸化的调控机制的分子分析奠定基础，并更好地理解kDNA表达中与生命周期相关的变化。这项研究的具体假设是，锥虫线粒体中的多聚腺苷酸化对功能性mRNA的稳定性至关重要，因此对线粒体功能至关重要。我们认为，多聚腺苷酸化是由动基体多聚（A）聚合酶（kPAP 1）作为多蛋白复合物的催化亚基完成的。KPAP 1已经在该提议的初步实验过程中通过与RNA尿苷酰转移酶（TUTases）的同源性被鉴定。我们的研究结果扩展了已知的TUTase样结构支架的作用，参与线粒体RNA加工的ATP特异性酶。我们建议：1.表征kPAP 1在非编辑、预编辑和编辑mRNA的多聚腺苷酸化中的作用。RNA干扰将用于下调前循环和血流形式的kPAP的表达，其对线粒体功能的意义将通过细胞活力、膜电位和RNA转录物的比较分析来解决。2.阐明锥虫线粒体中多聚腺苷酸化机制的蛋白质组成。将通过串联亲和色谱法分离含kPAP 1的复合物，检测酶活性，并通过质谱法进行分析。 叙述：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国军队中内脏利什曼病的发生，在美国也可以预期对有效的抗锥虫疗法的需求。现有的治疗方法往往是有毒和无效的，创造了发现新药的需求。靶向必需的寄生虫特异性酶，如我们的提案中首次描述的线粒体poly（A）聚合酶，是创造新一代杀锥虫剂的有希望的方法。然而，需要对聚腺苷酸化过程进行详细的分子分析，以验证这种酶作为药物靶点。

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.