Dissecting essential signaling pathways in apicomplexan parasites

7.项目概要/摘要 顶复门寄生虫是重要的人类病原体，并且引起从终生的 弓形虫无症状感染者约占全球人口的四分之一，达到近百万人 每年死于疟疾的人数。为了破解它们的生物学原理并治疗它们引起的疾病，我们必须 了解这些成功病原体的独特信号通路。钙依赖蛋白激酶 （CDPKs）是有吸引力的干预目标，因为它们在顶复门中是保守的， 从动物宿主的基因组中分离出来，这对寄生虫的生命周期至关重要。先前的工作表明， CDPKs调节T.弓形虫的生命周期，包括钙调节 运动所需的特殊细胞器的分泌。尽管我们已经确定了 T.我们对弓形虫的底物知之甚少，对弓形虫的后果更是知之甚少。 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。 这项拟议的研究将绘制由顶复门CDPKs调控的重要信号通路，并为他们提供信息。 作为治疗靶点的潜力。该提案的三个具体目标将涉及社区发展、知识产权和知识产权的不同方面 生物学，通过识别单个激酶的作用，表征它们调节的底物，以及 确定这些底物的功能。第一个目标是使用化学遗传学策略， 本申请人的目的是特异性抑制和研究两种CDPK在寄生虫生命周期中的功能，并将其扩展到 这一策略适用于激酶家族的其余四个成员。这些实验将使我们能够比较 细胞过程由T.刚地。第二个目标是利用我们的能力 标记和鉴定特异性寄生虫激酶的靶点，绘制先前两种CDPK的底物， 显示对于寄生虫进入和离开宿主细胞是必需的。最终的目标将使用定量质量 光谱和遗传操作指导的CDPK目标，我们已经确定和那些确定在 第二个目的是测量体内磷酸化的变化，并确定选定的CDPK的功能 目标的第二个和第三个目标将共同表征由CDPK调节的途径的组分， 并建立其基本功能的分子基础。 本研究的目的是绘制由顶复门CDPKs调控的重要信号网络， 作为治疗靶点的潜力。新发现的个别激酶的底物可能是新的 这些途径的组成部分。这是相关的，因为我们不知道约40%的顶复体的功能 蛋白质或它们参与的途径。此外，本研究还为比较 CDPK在顶复门中发挥作用，以揭示这种激酶家族如何调节不同的细胞行为。 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.