Dissecting the calcium dependent phosphorylation network of Toxoplasma gondii

剖析弓形虫的钙依赖性磷酸化网络

• 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
• 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

 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，是影响人类的最重要的传染原。由于普遍存在的耐药性、毒性和针对某些阶段缺乏活性，迫切需要新的抗apicomplexan药物。这些寄生虫中的钙信号传导作为潜在的药物靶标值得特别关注，因为它驱动许多重要事件，例如运动、入侵和流出，并且包含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，弓形虫 CDPK（TgCDPK3）是有效排出、分裂、钙稳态和体内慢性感染建立所必需的，大概是通过维持寄生虫发挥作用的正常生理状态来实现的。为了了解这些不同表型背后的机制，我们通过近系统范围的磷酸化蛋白质组学方法测量了野生型和 TgCDPK3 突变体寄生虫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，这些蛋白质以 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。作为一种补充方法，我们对相互作用蛋白进行了基于生物素化的筛选，并鉴定了 13 种推定的 TgCDPK3 相关蛋白，其中 7 种也被鉴定为在突变菌株的磷酸化蛋白质组中含量较低。我们假设 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。与这一观点一致，TgCDPK3 或磷酸酶钙调神经磷酸酶的化学抑制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裂解循环的“磷酸化程序”。为此，我们将： 1) 通过生成和表征每种 TgCDPK3 假定底物的突变寄生虫系并定义其细胞定位，确定十种 TgCDPK3 假定底物在裂解周期中的作用。 2) 鉴定磷酸化事件的序列以及所鉴定的 156 个磷酸位点网络中磷酸化的化学计量。我们将使用高度创新的靶向蛋白质组方法来实现这一点，该方法允许非常快速和高覆盖率的磷酸蛋白质组分析。使用各种突变株，我们还将确定作为网络本身一部分的几种蛋白质对信号级联的相对贡献。 3) 通过基因破坏钙依赖性磷酸酶钙调磷酸酶并使用两种互补的方法来定义其调节的蛋白质，从而确定钙依赖性磷酸酶钙调神经磷酸酶在流出过程中的功能和底物。结合起来，这些研究将深入了解磷酸化如何调节致病性寄生虫弓形虫的繁殖，这无疑将揭示寄生虫生物学中的漏洞，可用于开发新疗法。