Dissecting essential signaling pathways in apicomplexan parasites

描述(申请人提供)：Apicomplexan寄生虫是重要的人类病原体，可导致各种疾病，从约四分之一的世界人口终身无症状地感染弓形虫到每年近100万人死于疟疾。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的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，在动物宿主的基因组中缺失，对寄生虫的生命周期是必不可少的。先前的工作表明，CDPKs调节弓形虫生命周期中所需的各种过程，包括钙调节的特化细胞器的分泌，所需的 能动性。虽然我们已经确定了弓形虫磷酸化的关键酶，但我们对底物知之甚少，更不知道这些修饰对寄生虫进入、存活和从受感染的宿主细胞释放的后果。这项拟议的研究将绘制由apicplexan CDPKs调控的基本信号通路，并告知它们作为治疗靶点的潜力。这项申请的三个具体目标将解决CDPK生物学的不同方面，通过确定单个激酶的作用，表征它们调节的底物，并确定这些底物的功能。第一个目的是使用研究人员建立的化学遗传策略来特异性地抑制和研究寄生虫生命周期中两个CDPKs的功能，并将这一策略扩展到激酶家族的其余四个成员。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。第二个目的是利用我们标记和识别特定寄生虫激酶靶标的能力，绘制先前显示对寄生虫进出宿主细胞至关重要的CDPKs底物。最终目标将使用定量质谱学和遗传操作-在我们已经识别的CDPK靶标和第二个目标确定的那些靶标的指导下-测量体内磷酸化的变化，并确定选定的CDPK靶标的功能。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，并为其基本功能建立分子基础。这项研究的目的是绘制由apicplexan CDPKs调控的基本信号网络，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很可能是这些途径的新成分。这是相关的，因为我们不知道~40%的apicomplexan蛋白的功能或它们参与的途径。此外，这项研究为比较CDPK在顶端复合体中的功能提供了基础，以揭示该激酶家族是如何调节不同生物的行为的。

DESCRIPTION (provided by applicant): 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 application will address differen 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 investigator 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 tw 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 do not 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.