Dissecting essential signaling pathways in apicomplexan parasites

剖析顶端复门寄生虫的重要信号通路

• 批准号: 8737992
• 负责人: Sebastian Lourido
• 金额: $ 48.75万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-19 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8737992
• 关键词: Ablation; Address; Alleles; Animals; Behavior; Binding; Biological; Biology; Calcium; Categories; Cell physiology; Cells; Cessation of life; Cryptosporidium; Cysteine; Dental caries; Disease; Engineering; Enzymes; Family; Genetic; Genome; Goals; Human; Immunoprecipitation; Individual; Infection; Intervention; Label; Life; Life Cycle Stages; Malaria; Mammals; Maps; Mass Spectrum Analysis; Measures; Methods; Modeling; Modification; Molecular; National Institute of Allergy and Infectious Disease; Organelles; Organism; Parasites; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Plasmodium malariae; Population; Positioning Attribute; Process; Proteins; Radiolabeled; Reaction; Research Personnel; Resistance; Role; Signal Pathway; Signal Transduction; Site; Therapeutic; Toxoplasma gondii; Work; Yeasts; analog; base; biodefense; calcium-dependent protein kinase; cell motility; chemical genetics; genetic manipulation; improved; in vivo; inhibitor/antagonist; member; novel; novel strategies; pathogen; prevent; protein function; public health relevance; radiotracer; research study; therapeutic target

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.

描述（由申请人提供）：顶复门寄生虫是重要的人类病原体，引起的疾病范围从世界上约四分之一人口的弓形虫终身无症状感染到每年近百万人因疟疾死亡。为了破译它们的生物学特性并治疗它们引起的疾病，我们必须了解这些成功病原体所独有的信号通路。钙依赖性蛋白激酶（CDPK）是有吸引力的干预靶点，因为它们在顶复门动物中是保守的，在动物宿主的基因组中不存在，并且对于寄生虫的生命周期至关重要。先前的研究表明，CDPKs 调节弓形虫生命周期中必需的各种过程，包括钙调节的特殊细胞器的分泌。 动力。尽管我们已经确定了弓形虫中负责磷酸化的关键酶，但我们对底物知之甚少，更不了解这些修饰对寄生虫进入、存活和从受感染宿主细胞中释放的影响。拟议的研究将绘制由 apicomplexan CDPK 调节的重要信号通路，并揭示它们作为治疗靶点的潜力。该应用的三个具体目标将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来解决 CDPK 生物学的不同方面。第一个目标是使用研究人员建立的化学遗传策略来特异性抑制和研究寄生虫生命周期中两个 CDPK 的功能，并将该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 CDPK 调节的细胞过程。第二个目标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入和离开宿主细胞至关重要的 tw CDPK 底物图谱。最终目标将使用定量质谱和基因操作，以我们已经确定的 CDPK 靶标和第二个目标中确定的 CDPK 靶标为指导，测量体内磷酸化变化并确定所选 CDPK 靶标的功能。第二个和第三个目标将共同描述 CDPK 调节途径的组成部分，并为其基本功能奠定分子基础。本研究的目的是绘制由 apicomplexan CDPK 调节的重要信号网络，并揭示其作为治疗靶点的潜力。新发现的单个激酶的底物可能是这些途径的新成分。这是相关的，因为我们不知道约 40% 的 apicomplexan 蛋白的功能或它们参与的途径。此外，这项研究为比较不同apicomplexans的CDPK功能提供了基础，以揭示该激酶家族如何调节不同生物体的行为。