Dissecting the calcium dependent phosphorylation network of Toxoplasma gondii

剖析弓形虫的钙依赖性磷酸化网络

基本信息

批准号：
9085774
负责人：
Gustavo A Arrizabalaga
金额：
$ 51.18万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-01 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9085774
关键词：
Acquired Immunodeficiency Syndrome Affect Amino Acids Apicomplexa Attention Biology Biotinylation Brain Calcineurin Calcium Calcium Signaling Cell Culture Techniques Cells Cessation of life Chemicals Chronic Complement Cyclosporine Cyst Development Disease Drug Targeting Drug resistance Enzymes Equilibrium Event Family Genes Goals Homeostasis Human Immunocompromised Host Infection Infectious Agent Ions Knock-out Laboratories Lead Learning Life Lytic Phase Mammalian Cell Mass Spectrum Analysis Measures Metabolism Methods Monitor Parasite Control Parasites Pathogenesis Patients Peptides Pharmaceutical Preparations Phenotype Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Physiological Plasmodium falciparum Play Population Process Protein Dephosphorylation Proteins Proteomics Reaction Regulation Research Resources Role Signal Transduction Signaling Protein Stable Isotope Labeling Staging System Testing Time Toxic effect Toxoplasma Toxoplasma gondii Toxoplasmosis Work base calcineurin phosphatase calcium-dependent protein kinase cell motility combat in vivo inhibitor/antagonist innovation member mutant novel therapeutics obligate intracellular parasite phosphatase inhibitor phosphoproteomics programs protein protein interaction public health relevance stoichiometry transmission process

项目摘要

DESCRIPTION (provided by applicant): Some of the intracellular parasites of the phylum Apicomplexa, such as Plasmodium falciparum and Toxoplasma gondii, are among the most important infectious agents affecting humans. Due to rampant drug resistance, toxicity and lack of activity against certain stages there is a dire need for new anti-apicomplexan drugs. Calcium signaling in these parasites deserves special attention as a potential drug target as it drives many essential events such as motility, invasion and egress and it includes proteins not found in mammalian cells, such as the family of calcium dependent protein kinases (CDPKs). We recently discovered that a particular Toxoplasma CDPK, TgCDPK3, is required for efficient egress, division, calcium homeostasis and establishment of a chronic infection in vivo, presumably by maintaining the normal physiological state in which the parasite functions. To understand the mechanisms behind these various phenotypes we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant parasites through a near system-wide phosphoproteomic approach. This analysis revealed 156 peptides representing 106 proteins that are phosphorylated in a TgCDPK3 dependent manner, with many of them related to motility, ion-homeostasis, and metabolism. As a complementary approach, we performed a biotinylation-based screen for interacting proteins and identified 13 putative TgCDPK3-associated proteins, of which 7 were also identified as less abundant in the phosphoproteome of the mutant strain. We hypothesize that regulation of the phosphorylation state of a specific network of proteins by enzymes such as TgCDPK3 and calcium dependent phosphatases, is essential for the completion of Toxoplasma's lytic cycle. Consistent with this idea chemical inhibition of either TgCDPK3 or the phosphatase calcineurin disrupts parasite exit from the host-cell. The main goal of this proposal is to characterize what we refer to as the "phospho-program" of the lytic cycle. To do this we will: 1) Determine the role of ten TgCDPK3 putative substrates during the lytic cycle by generating and characterizing mutant parasite lines of each and defining their cellular localization. 2) Identify the sequence of phosphorylation events and the stoichiometry of phosphorylation in the identified network of 156 phosphosites. We will do this using a highly innovative targeted-proteomic approach that allows very rapid and high coverage phosphoproteome analysis. Using various mutant strains we will also determine the relative contribution of several proteins that are part of the network themselves on the signaling cascade. 3) Determine the function and substrates of the calcium-dependent phosphatase calcineurin during egress by genetically disrupting it and using two complimentary approaches to define the proteins it regulates. In conjunction, these studies will provide an in depth understanding of how phosphorylation regulates the propagation of the pathogenic parasite Toxoplasma, which would undoubtedly reveal vulnerabilities in the parasite's biology that can exploited for the development of new therapies.

描述（由申请人提供）：顶复门的一些细胞内寄生虫，例如恶性疟原虫和弓形虫，由于其猖獗的耐药性、毒性和针对某些阶段缺乏活性，是影响人类的最重要的传染原。这些寄生虫中迫切需要新的抗 apicomplexan 药物，作为潜在的药物靶点值得特别关注，因为它驱动许多重要事件，例如它包括哺乳动物细胞中未发现的蛋白质，例如钙依赖性蛋白激酶 (CDPK) 家族，我们最近发现一种特殊的弓形虫 CDPK（TgCDPK3）是有效的流出、分裂、钙稳态和代谢所必需的。体内慢性感染的建立，大概是通过维持寄生虫发挥作用的正常生理状态来了解这些不同表型背后的机制，我们测量了野生型和 TgCDPK3 中的相对磷酸化位点使用情况。该分析揭示了代表 106 种蛋白质的 156 种肽，它们以 TgCDPK3 依赖性方式磷酸化，其中许多与运动性、离子稳态和代谢有关。基于生物素化的相互作用蛋白筛选，鉴定出 13 种推定的 TgCDPK3 相关蛋白，其中 7 种也被鉴定为在我们发现，TgCDPK3 和钙依赖性磷酸酶等酶对特定蛋白质网络的磷酸化状态的调节对于弓形虫裂解周期的完成至关重要，这与 TgCDPK3 或 TgCDPK3 的化学抑制的想法一致。磷酸酶钙调神经磷酸酶会破坏寄生虫从宿主细胞中的退出。该提案的主要目标是描述我们所说的“寄生虫”。为了做到这一点，我们将： 1) 通过生成和表征每种突变寄生虫系并定义其细胞定位来确定十种 TgCDPK3 假定底物的作用 2) 识别序列。我们将使用高度创新的靶向蛋白质组学方法来实现这一点，该方法可以非常快速且快速地进行。高覆盖度磷酸蛋白质组分析。我们还将使用各种突变菌株来确定属于网络本身的几种蛋白质对信号级联的相对贡献。 3) 通过基因破坏来确定钙依赖性磷酸酶钙调神经磷酸酶的功能和底物。结合使用两种互补的方法来定义其调节的蛋白质，这些研究将深入了解磷酸化如何调节病原寄生虫的繁殖。弓形虫，这无疑将揭示寄生虫生物学中的弱点，可用于开发新疗法。