Studies of DNA Licensing in Apicomplexa Parasites

顶复门寄生虫 DNA 许可的研究

• 批准号: 9196820
• 负责人: Michael W White
• 金额: $ 53.85万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-14 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9196820
• 关键词: Alleles; Animals; Apicomplexa; Biology; CDK2 gene; Cell Cycle; Cell Cycle Progression; Cell Nucleus; Cell division; Cells; Cessation of life; Chemicals; Chromosome Segregation; Chromosomes; Clinical Treatment; Complex; Cyclin-Dependent Kinases; Cyclins; Cytolysis; DNA; DNA biosynthesis; Daughter; Disease; Drug resistance; Drug usage; Enabling Factors; Eukaryota; Future; Genes; Genetic; Glycylglycine; Goals; Growth; High temperature of physical object; Host Defense; Human; Infection; Knowledge; Lead; Licensing; Licensing Factor; Longevity; Malaria; Molecular; Mutation; Nuclear; Nuclear Protein; Orthologous Gene; Parasite Control; Parasites; Pathway interactions; Pharmacotherapy; Phenotype; Phosphorylation; Phosphotransferases; Point Mutation; Process; Proteins; Proteomics; Protozoa; Role; S Phase; Staging; Temperature; Testing; Therapeutic Intervention; Topoisomerase II; Toxoplasma; Toxoplasma gondii; Ubiquitination; Yeasts; asexual; base; chromosome replication; combat; effective therapy; flexibility; helicase; mutant; novel; parasite invasion; pathogen; pressure; research study; temperature sensitive mutant; ubiquitin-protein ligase; yeast two hybrid system

Existing therapies, particularly against malaria, are under constant pressure from acquired parasite drug resistance requiring a continuing search for new treatments. The peculiar proliferative cycles of Apicomplexa parasites differ substantially from the hosts they inhabit and should offer fertile ground to supply an active pipeline of new targets. To fulfill this promise, we need a better understanding of the unique structural and molecular features of parasite cell division. Apicomplexan proliferation has adapted to different host cells using chromosome replication cycles that can vary in the scale of nuclear reduplication from a few to hundreds of nuclei produced per day, which is unparalleled cell cycle flexibility. How fidelity is preserved through variable rounds of chromosome replication is a major mystery of Apicomplexa biology as many known regulators of DNA replication in multicellular eukaryotes are missing in these parasites. Further, the basic checkpoint mechanisms that regulate the cell cycle transitions are also poorly understood. It is not known what controls G1 to S phase commitment, S phase progression, chromosome segregation or what controls allow the parasite to forgo budding in one type of chromosome cycle but not in another during the processes of schizogony and endopolygeny. In this application we will investigate several Toxoplasma mutants that carry lethal point mutations in proteins essential for proper chromosome replication and segregation. When shifted to high temperature these mutants all suffer very similar disruptions in chromosome replication. In Aim 1, we will define the molecular function of a novel RING protein (ECR1, essential for chromosome replication 1) in regulating chromosome replication and segregation. We will determine whether ECR1 is a divergent E3 ligase and compare the molecular features of this mechanism in controlling DNA replication to the known chromosome licensing factor Topo-II. We will also investigate an alternate role for ECR1 in regulating the tachyzoite cell cycle. ECR1 forms a complex with a Toxoplasma ortholog of human cyclin-dependent kinase 2 (TgCDK2). The ECR1/TgCDK2 complex appears in the centrocone and then leaves this compartment to become exclusively nuclear during S phase. We will characterize the molecular basis for this interaction and determine whether this partnership is required for ECR1 function. We will also determine the basic features of the TgCDK2 mechanism including whether it requires a cyclin for function and identify the protein substrates of TgCDK2 in order to understand how this kinase mechanism regulates the tachyzoite S phase progression. In Aim 2, we will investigate two chemical mutants harboring two other defective ECR factors (ECR2 and 3) that also cause uncontrolled DNA synthesis when mutant parasites are grown at high temperature. ECR2 and ECR3 are unknown proteins conserved only within Apicomplexa genetic lineages. The discovery of apicomplexan-specific factors essential for chromosome replication supports our central hypothesis that these ancient parasites have evolved unique molecular mechanisms to regulate asexual stage proliferation.

现有的治疗方法，特别是针对疟疾的治疗方法，不断受到来自获得性寄生虫药物的压力。 需要不断寻找新的治疗方法。顶复门特有的增殖周期 寄生虫与它们栖息的宿主有很大的不同，应该提供肥沃的土壤， 新目标的管道。为了实现这一承诺，我们需要更好地了解独特的结构和 寄生虫细胞分裂的分子特征顶复门的增殖已经适应了不同的宿主细胞， 染色体复制周期，其核复制的规模可以从几个到数百个不等。 细胞周期的灵活性无与伦比。如何通过变量保持保真度 染色体复制的循环是顶复门生物学的一个主要谜团，因为许多已知的染色体复制的调节因子， 多细胞真核生物中的DNA复制在这些寄生虫中缺失。此外，基本检查点 调节细胞周期转换的机制也知之甚少。不知道是什么控制了 G1至S期定型、S期进展、染色体分离或何种控制措施允许寄生虫 在有性生殖过程中，在一种类型的染色体周期中放弃出芽，而在另一种类型的染色体周期中不放弃出芽， 内多源性在这个应用程序中，我们将调查几个弓形虫突变体，携带致死点 染色体复制和分离所必需的蛋白质突变。当切换到高电平时 这些突变体都遭受非常相似的染色体复制中断。在目标1中，我们 定义了一种新的RING蛋白（ECR 1，染色体复制所必需的1）的分子功能， 调节染色体复制和分离。我们将确定ECR 1是否是一个趋异的E3连接酶 并将这种控制DNA复制机制的分子特征与已知的 染色体许可因子Topo-II。我们还将研究ECR 1在调节细胞凋亡中的替代作用。 速殖子细胞周期ECR 1与人细胞周期蛋白依赖性激酶2的弓形虫直系同源物形成复合物 （TgCDK2）。ECR 1/TgCDK 2复合物出现在中心锥中，然后离开该隔室， 在S阶段变成纯核的。我们将描述这种相互作用的分子基础， 确定ECR 1功能是否需要此伙伴关系。我们还将确定 TgCDK 2的机制，包括它是否需要细胞周期蛋白的功能，并确定蛋白质底物， TgCDK 2，以了解这种激酶机制如何调节速殖子S期进展。在 目的2，我们将研究两种含有另外两种缺陷型ECR因子（ECR 2和3）的化学突变体， 当突变寄生虫在高温下生长时，也会导致不受控制的DNA合成。教统会第二号报告书及 ECR 3是仅在顶复门遗传谱系中保守的未知蛋白。的发现 染色体复制所必需的顶复体特异性因子支持我们的中心假设， 古老的寄生虫已经进化出独特的分子机制来调节无性阶段的增殖。