Cryptosporidium parvum DNA replication proteins

描述（由申请人提供）： 隐孢子虫引起艾滋病患者的机会性感染之一，目前还没有治疗方法。C.小孢子由几个不同的细胞周期组成，称为孢子生殖、部分生殖、配子生殖和卵囊形成。在孢子生殖和部分生殖期间，寄生虫在每个细胞周期中进行增殖以形成4至8个子细胞，这与其宿主中的重复细胞周期不同，并表明一种独特的机制可能调节顶复门的增殖。复制蛋白A（RPA）是真核生物单链DNA（ssDNA）结合蛋白，由3个亚基组成，在DNA复制、修复和重组中发挥多种作用。我们以前发现了一种短型C。与人的RPA大亚基（约70 kDa）不同的微小RPA大亚基（CpRPA 1A，54 kDa）。最近，第二个RPA 1同源物（CpRPA 1 B）从寄生虫中鉴定出来，与CpRPA 1A不同。由于人类只有一种RPA 1蛋白，Cp中存在两种不同的CpRPA 1A和1B蛋白，再加上CpRPA 1A和1B都缺乏N-末端调节结构域的事实，使我们假设C.小孢子虫不仅具有独特的DNA复制蛋白，而且可能在DNA代谢的调控上与宿主不同。由于所有生物体的生长和发育都依赖于每个细胞周期中DNA复制的忠实复制，寄生虫中特有的DNA复制机制可能成为抗隐孢子虫病的新药物靶标。我们的长期目标是阐明Cp复制蛋白的结构和功能与独特的寄生虫细胞和生命周期的化疗探索。该项目的具体目标是研究CpRPA 1A和CpRPA 1B基因的表达，翻译后加工和功能特性，使用蛋白质组学，代谢标记，分子工程，免疫标记，共聚焦和电子显微镜，以及弓形虫转染系统。RPA蛋白已成为有前途的针对各种癌症的化疗靶点。人与C. parvum RPA蛋白及其功能支持寄生虫RPA可作为潜在药物靶标的观点。这些目标的完成不仅可以证明或反驳我们的假说，而且为靶向寄生虫独特的DNA机制的药物发现奠定了新的基础。

DESCRIPTION (provided by applicant): Cryptosporidium parvum causes one of the opportunistic infections among AIDS patients for which no treatment is yet available. The development of C. parvum consists of several distinct cell cycles referred to as sporogony, merogony, gametogony and oocyst formation. During sporogony and merogony, parasites undergo multiplication to form 4 to 8 daughter cells in each cell cycle, which differs from the duplicative cell cycle in its hosts, and suggests that a unique mechanism may regulate apicomplexan multiplications. Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA)-binding protein, consisting of 3 subunits and playing multiple roles in the DNA replication, repair, and recombination. We have previously discovered a short-type C. parvum RPA large subunit (CpRPA1A, 54 kDa) that differs from that of humans (approximately 70 kDa). More recently, a second RPA1 homologue (CpRPA1 B) was identified from the parasite that differs from CpRPA1A. Since humans have only one RPA1 protein, the presence of two distinct CpRPA1A & 1B proteins in Cp, together with the fact that both CpRPA1A & 1B lack the N-terminal regulatory domain, lead us to hypothesize that C. parvum not only possesses unique DNA replication proteins, but possibly also differs from its host in the regulation of DNA metabolism. Since the growth and development of all organisms depend on the faithful replication of DNA replication in each cell cycle, the peculiar DNA replication machinery in the parasite may be pursued as a novel drug target against cryptosporidiosis. Our long-term goal is to elucidate the structure and function of Cp replication proteins associated with the unique parasite cell and life cycles for chemotherapeutic exploration. The specific aim for the project is to investigate the expression, posttranslational processing and functional properties of CpRPA1A and CpRPA1B genes using proteomics, metabolic labeling, molecular engineering, immunolabeling, confocal and electron microscopy, and a Toxoplasma gondii transfection system. RPA proteins have been promising chemotherapeutic targets against various cancers. The apparent differences between human and C. parvum RPA proteins and their functions support the notion that the parasite RPA may serve as a potential drug target. The completion of these aims will not only prove or refute our hypothesis, but also establish a new foundation for the drug discovery targeting the parasite unique DNA mechanism machinery.