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.

现有的疗法，特别是针对疟疾的疗法，一直受到来自获得性寄生虫药物的压力 耐药性需要不断寻找新的治疗方法。 Apicomplexa 独特的增殖周期 寄生虫与其栖息的宿主有很大不同，应该提供肥沃的土壤来提供活性 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。顶复体增殖已适应不同的宿主细胞 染色体复制周期的核重复规模可以从几个到数百个不等 每天产生细胞核，这是细胞周期无与伦比的灵活性。如何通过变量保持保真度 染色体复制的轮次是顶复门生物学的一个主要谜团，因为许多已知的调节因子 这些寄生虫缺乏多细胞真核生物中的 DNA 复制。此外，基本检查点 调节细胞周期转变的机制也知之甚少。尚不清楚是什么控制着 G1 到 S 期的承诺、S 期进展、染色体分离或哪些控制允许寄生虫 在分裂过程中放弃一种染色体周期的出芽，但不放弃另一种染色体周期的出芽 内多生。在此应用中，我们将研究几种携带致死点的弓形虫突变体 对于染色体正确复制和分离至关重要的蛋白质突变。当切换到高电平时 在不同温度下，这些突变体都遭受了非常相似的染色体复制破坏。在目标 1 中，我们将 定义新型 RING 蛋白（ECR1，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 ECR1 是否是分歧的 E3 连接酶 并将这种控制 DNA 复制的机制的分子特征与已知的进行比较 染色体许可因子Topo-II。我们还将研究 ECR1 在调节 速殖子细胞周期。 ECR1 与人细胞周期蛋白依赖性激酶 2 的弓形虫同源物形成复合物 （TgCDK2）。 ECR1/TgCDK2 复合物出现在中心锥中，然后离开该隔室 在 S 阶段完全成为核。我们将描述这种相互作用的分子基础，并 确定 ECR1 功能是否需要这种伙伴关系。我们还将确定的基本特征 TgCDK2 机制，包括是否需要细胞周期蛋白来发挥功能并识别其蛋白质底物 TgCDK2，以了解该激酶机制如何调节速殖子 S 期进展。在 目标 2，我们将研究两种含有另外两个有缺陷的 ECR 因子（ECR2 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 DNA 合成不受控制。 ECR2 和 ECR3 是仅在顶复门遗传谱系中保守的未知蛋白质。的发现 染色体复制所必需的顶端复合体特异性因子支持我们的中心假设，即这些因子 古代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。