Dissecting essential signaling pathways in apicomplexan parasites

7.项目摘要/摘要 Apicomplexan寄生虫是重要的人类病原体，可导致终生疾病 弓形虫无症状感染在约四分之一的世界人口中达到近100万 每年死于疟疾的人数。为了破译它们的生物学并治疗它们引起的疾病，我们必须 了解这些成功病原体特有的信号通路。钙依赖蛋白激酶 CDPKs是有吸引力的干预靶点，因为它们在顶端复合体中保守，不存在 从动物宿主的基因组中分离出来，对寄生虫的生命周期至关重要。先前的研究表明， CDPKs调控弓形虫生命周期所必需的各种过程，包括钙调节的 运动所需的特殊细胞器的分泌。尽管我们已经确定了负责的关键酶 对于弓形虫的磷酸化，我们对底物知之甚少，更不知道 这些修饰用于寄生虫进入、存活和从受感染的宿主细胞释放。 拟议的研究将绘制由apicplexan CDPKs调控的基本信号通路，并告知他们 有可能成为治疗靶点。这项提案的三个具体目标将涉及CDPK的不同方面 生物学，通过确定单个激酶的作用，表征它们调节的底物，以及 确定这些底物的功能。第一个目标是使用由 申请者专门抑制和研究两个CDPKs在寄生虫生命周期中的功能，并将其延长 策略传递给剩下的四个蛋白激酶家族成员。这些实验将使我们能够比较 弓形虫中每个保守的CDPKs调控的细胞过程。第二个目标是利用我们的能力 标记和鉴定特定寄生虫蛋白激酶的靶标，映射两个CDPKs的底物 被证明是寄生虫进出宿主细胞所必需的。最终目标将使用定量质量 光谱和遗传操作-由我们已经识别的CDPK靶标和 第二个目的-测量体内磷酸化的变化并确定选定的CDPK的功能 目标。第二个和第三个目标将共同描述由CDPKs调控的通路的组成部分， 并为其基本功能奠定了分子基础。 这项研究的目标是绘制由apicplexan CDPKs调控的基本信令网络，并告知 它们作为治疗靶点的潜力。新发现的单个激酶的底物可能是新的 这些路径的组成部分。这是相关的，因为我们不知道~40%的顶复合体的功能 蛋白质或它们参与的途径。此外，本研究还提供了比较的基础。 CDPK在顶端复合体中发挥作用，以揭示该激酶家族如何调节不同的 有机体。

7. PROJECT SUMMARY/ ABSTRACT Apicomplexan parasites are important human pathogens, and cause diseases ranging from life-long asymptomatic infections with Toxoplasma gondii in about a quarter of the world's population to nearly a million deaths annually due to malaria. To decipher their biology and treat the diseases they cause, we must understand the signaling pathways unique to these successful pathogens. Calcium-dependent protein kinases (CDPKs) are attractive targets for intervention because they are conserved among apicomplexans, absent from the genomes of their animal hosts, and essential for the parasite life cycle. Prior work has shown that CDPKs regulate various processes necessary during the T. gondii life cycle, including the calcium-regulated secretion of specialized organelles required for motility. Although we have identified key enzymes responsible for phosphorylation in T. gondii, we know little about the substrates, and even less about the consequences of these modifications for parasite entry, survival and release from the infected host cell. The proposed study will map essential signaling pathways regulated by apicomplexan CDPKs and inform their potential as therapeutic targets. The three specific aims of this proposal will address different aspects of CDPK biology, by identifying the role of individual kinases, characterizing the substrates they regulate, and determining the function of these substrates. The first aim uses a chemical-genetic strategy established by the applicant to specifically inhibit and study the function of two CDPKs in the parasite life cycle, and extends this strategy to the four remaining members of the kinase family. These experiments will allow us to compare the cellular processes regulated by each of the conserved CDPKs in T. gondii. The second aim exploits our ability to label and identify the targets of specific parasite kinases, to map the substrates of two CDPKs previously shown to be essential for parasite entry and exit from host cells. The final aim will use quantitative mass spectrometry and genetic manipulation-guided by CDPK targets we already identified and those identified in the second aim-to measure phosphorylation changes in vivo and determine the function of selected CDPK targets. Together the second and third aims will characterize components of the pathways regulated by CDPKs, and establish the molecular basis for their essential function. The goal of this study is to map essential signaling networks regulated by apicomplexan CDPKs and inform their potential as therapeutic targets. Newly identified substrates of individual kinases are likely novel components of these pathways. This is relevant because we don't know the function of ~40% of apicomplexan proteins or the pathways in which they participate. Furthermore, this study provides the basis for comparing CDPK functions across apicomplexans, to uncover how this kinase family regulates the behavior of different organisms.