Structural Analysis of DNA Replication Machinery of P. falciparum

描述（由申请人提供）：疟疾是世界上导致疾病和死亡的主要原因。每年有3亿多人受到感染，造成1至300万人死亡。除了引起致残性发热和寒战外，疟疾感染还与伯基特淋巴瘤有因果关系，在流行地区，伯基特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，几乎导致所有相关死亡。恶性疟原虫含有一种称为顶质体的重要细胞器，当其功能或复制受到抑制时，可杀死与恶性疟原虫有关的寄生虫。我建议将PfPrex作为一种有前景的药物靶点进行研究，PfPrex被认为是恶性疟原虫顶质体的复制机制。PfPrex具有良好的进化保守性，具有引物酶、解旋酶、核酸外切酶和聚合酶活性。它在体内被蛋白质水解成熟成几个较小的结构域，这让人想起病毒的多蛋白策略。抑制这些酶活性，或抑制PfPrex的成熟，可能会抑制顶质体的复制，进而导致寄生虫的死亡。这些建议的目的是阐明顶体功能的机制，并确定PfPrex的基本元素作为治疗疟疾感染和预防地方性伯基特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的引物酶结构域的三维结构是什么？(2) PfPrex在体内起什么作用？首先，我从基因组DNA中克隆了PfPrex的功能性引物酶结构域，从大肠杆菌中过度表达和纯化了这种蛋白质，并开始对其进行生物化学表征。现在，我将继续通过体外实验检测PfPrex引物酶，并使用x射线晶体学确定其三维结构。其次，使用我开发的针对PfPrex TOPRIM结构域的多克隆抗体，我将检查PfPrex的蛋白水解裂解是否需要其有效功能，以及在寄生虫的整个无性生命周期中鉴定PfPrex在体内的成熟功能形式。公共卫生相关性：在引起疟疾的寄生虫种类中，恶性疟原虫几乎导致所有相关死亡，并与地方性伯基特淋巴瘤有因果关系。我打算研究一种叫做PfPrex的蛋白质，这种寄生虫可能需要它来生长和生存。利用关于PfPrex如何在寄生虫中组装，功能和加工的信息，我将确定特定的新药靶点来抑制其功能，这可能会杀死寄生虫，治疗疟疾感染，并预防地方性伯基特淋巴瘤。

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. PUBLIC HEALTH RELEVANCE: Of the parasite species that cause malaria, Plasmodium falciparum causes nearly all associated deaths, and also is causally-associated with endemic Burkitt's Lymphoma. I intend to study a protein, called PfPrex, which this parasite likely needs to grow and survive. Using information gained about how PfPrex assembles, functions and is processed in the parasite, I will identify specific new drug targets to inhibit its functions, which will likely kill the parasite, treat malarial infection, and prevent endemic Burkitt's Lymphoma.