Cryptosporidium parvum DNA replication proteins

小隐孢子虫 DNA 复制蛋白

基本信息

批准号：
6751711
负责人：
GUAN ZHU
金额：
$ 21.83万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-06-01 至 2006-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6751711
关键词：
Cryptosporidium DNA binding protein DNA replication SDS polyacrylamide gel electrophoresis Toxoplasma gondii autoradiography confocal scanning microscopy electron microscopy gene expression immunoprecipitation nucleic acid metabolism parasitism posttranslational modifications protein structure function proteomics transfection

项目摘要

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.

描述（由申请人提供）：小隐孢子虫是艾滋病患者中的一种机会性感染，目前尚无治疗方法。小隐孢子虫的发育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。在孢子生殖和子生殖期间，寄生虫在每个细胞周期中进行增殖，形成4至8个子细胞，这与其宿主中的复制细胞周期不同，并且表明独特的机制可能调节顶复体增殖。复制蛋白 A (RPA) 是一种真核单链 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。我们之前发现了一个短型 C. parvum RPA 大亚基（CpRPA1A，54 kDa），与人类的 RPA 大亚基（约 70 kDa）不同。最近，从与 CpRPA1A 不同的寄生虫中鉴定出第二个 RPA1 同源物 (CpRPA1 B)。由于人类只有一种 RPA1 蛋白，因此 Cp 中存在两种不同的 CpRPA1A 和 1B 蛋白，再加上 CpRPA1A 和 1B 都缺乏 N 末端调节结构域，因此我们推测 C. parvum 不仅拥有独特的 DNA复制蛋白，但在 DNA 代谢的调节方面也可能与其宿主不同。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，因此寄生虫中独特的 DNA 复制机制可以作为对抗隐孢子虫病的新药物靶标。我们的长期目标是阐明与独特的寄生虫细胞和生命周期相关的 Cp 复制蛋白的结构和功能，以进行化疗探索。该项目的具体目标是利用蛋白质组学、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研究 CpRPA1A 和 CpRPA1B 基因的表达、翻译后加工和功能特性。 RPA 蛋白一直是针对各种癌症的有希望的化疗靶点。人类和微小隐球菌 RPA 蛋白及其功能之间的明显差异支持了寄生虫 RPA 可能作为潜在药物靶点的观点。这些目标的完成不仅将证明或反驳我们的假设，还将为针对寄生虫独特DNA机制的药物发现奠定新的基础。