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）：Apicomplexan寄生虫是重要的人类病原体，并引起疾病，从终身无症状感染伴有弓形虫弓形虫的疾病，大约四分之一的人口，每年由于疟疾而导致的近一百万人死亡。为了破译其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所特有的信号通路。钙依赖性的蛋白激酶（CDPK）是干预的有吸引力的靶标，因为它们在Apicomplexans中是保守的，其动物宿主的基因组缺乏，对于寄生虫生命周期至关重要。先前的工作表明，CDPK调节了T. gondii生命周期中必要的各种过程，包括钙调节的分泌。 运动。尽管我们已经鉴定出负责T. gondii中磷酸化的关键酶，但我们对底物知之甚少，甚至对这些修饰对寄生虫进入，存活和从感染宿主细胞中释放的后果的了解甚至更少。拟议的研究将绘制由Apicomplexan CDPK调节的基本信号通路，并告知其作为治疗靶标的潜力。该应用程序的三个特定目的将通过识别单个激酶的作用，表征其调节的底物并确定这些底物的功能来解决CDPK生物学的不同方面。第一个目的使用研究者建立的化学遗传策略来专门抑制和研究两个CDPK在寄生虫生命周期中的功能，并将这种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T. gondii中每个保守的CDPK调节的细胞过程。第二个目标利用了我们标记和识别特定寄生虫激酶的靶标的能力，以绘制先前显示的TW CDPK的底物对于寄生虫进入和从宿主细胞退出至关重要。最终目标将使用我们已经确定的CDPK靶标和第二个目标测量体内磷酸化变化的CDPK靶标引导的定量质谱和遗传操纵，并确定所选CDPK靶标的功能。第二和第三目标将表征由CDPK调节的途径的组成部分，并为其基本功能建立分子基础。这项研究的目的是绘制由APICOMPLEXAN CDPK调节的基本信号网络，并告知其作为治疗靶标的潜力。新鉴定的单个激酶的底物可能是这些途径的新成分。这很重要，因为我们不知道〜40％的Apicomplexan蛋白或它们参与的途径的功能。此外，这项研究为比较跨Apicomplexans的CDPK功能提供了基础，以发现该激酶家族如何调节不同生物的行为。