Structural Analysis of DNA Replication Machinery of P. falciparum

描述(申请人提供)：疟疾是世界上导致疾病和死亡的主要原因。每年有超过3亿人感染，每年造成100-300万人死亡。除了引起发热和寒战外，疟疾感染还与Burkitt淋巴瘤有因果关系，在流行地区，这种淋巴瘤占儿童恶性疾病的74%。在导致疟疾的物种中，恶性疟原虫是毒性最强的，并导致几乎所有相关的死亡。恶性疟原虫含有一种被称为顶体的重要细胞器，当其功能或复制被抑制时，就会杀死与恶性疟原虫相关的寄生虫。我建议研究PfPrex，这是恶性疟原虫质外体可能的复制机制，作为一个有希望的药物靶点。PfPrex在进化上是保守的，并显示出底物酶、解旋酶、核酸外切酶和聚合酶活性。它在体内被蛋白质降解成熟为几个较小的结构域，这让人想起病毒多蛋白策略。抑制这些酶的活性，或抑制PfPrex的成熟，可能会抑制质外体的复制，进而导致寄生虫的死亡。这些提议的目的是阐明质外体功能的机制，并确定PfPrex的基本成分作为治疗疟疾感染和预防地方性Burkitt淋巴瘤的新药的靶点。我建议通过解决以下两个问题来实现这一点：(1)PfPrex的启动子结构域的三维结构是什么？以及(2)PfPrex在体内的作用形式是什么？首先，我从基因组DNA中克隆了PfPrex的功能起始酶结构域，在大肠杆菌中高效表达和纯化了该蛋白，并对其进行了生化鉴定。我现在将继续通过体外测试来检测PfPrex Primase，并使用X射线结晶学来确定其三维结构。其次，使用我开发的针对PfPrex的TOPRIM结构域的多克隆抗体，我将检查PfPrex的高效功能是否需要PfPrex的蛋白水解性切割，以及在寄生虫的无性生命周期中鉴定体内成熟的、功能性的PfPrex形式。

DESCRIPTION (provided by applicant): Malaria is a leading cause of disease and death in the world. Over 300 million people are infected annually, causing 1-3 million deaths per year. In addition to causing incapacitating fever and chills, malarial infection is causally-associated with Burkitt's Lymphoma, which accounts for ~74 percent of childhood malignant disorders in endemic regions. Of the species that cause malaria, Plasmodium falciparum is the most virulent, and causes nearly all associated deaths. P. falciparum contains an essential organelle, termed the apicoplast, that when its functions or replication are inhibited kills parasites related to P. falciparum. I propose to investigate PfPrex, the putative replicative machinery of P. falciparum's apicoplast, as a promising drug target. PfPrex is evolutionarily well-conserved and has demonstrated primase, helicase, exonuclease and polymerase activities. It is proteolytically matured into several smaller domains in vivo, which is reminiscent of viral poly-protein strategies. Inhibition of these enzymatic activities, or of the maturation of PfPrex, will likely inhibit replication of the apicoplast, and in turn cause the death of the parasite. The goals of these proposed aims is to illuminate mechanisms of apicoplast function, and to identify essential elements of PfPrex as targets for novel drugs to treat malarial infection and prevent endemic Burkitt's Lymphoma. I propose to accomplish this by addressing the following two questions: (1) What is the three-dimensional structure of the primase domain of PfPrex? and (2) What forms of PfPrex function in vivo? First, I have cloned the functional primase domain of PfPrex from genomic DNA, over-expressed and purified this protein from E. coli, and begun to characterize it biochemically. I will now continue to examine PfPrex primase through in vitro assays, and determine its three-dimensional structure using x-ray crystallography. Second, using polyclonal antibodies that I have developed against PfPrex's TOPRIM domain, I will examine if proteolytic cleavage of PfPrex is required for its efficient function, as well as to identify the mature, functional forms of PfPrex in vivo throughout the asexual life cycle of the parasite.