Dissecting the calcium dependent phosphorylation network of Toxoplasma gondii

 描述(申请人提供)：一些尖端复合体的细胞内寄生虫，如恶性疟原虫和弓形虫，是影响人类的最重要的感染性病原体之一。由于广泛的耐药性、毒性和对某些阶段缺乏活性，迫切需要新的抗心尖复合体药物。这些寄生虫中的钙信号作为一个潜在的药物靶点值得特别关注，因为它驱动许多重要的事件，如运动、入侵和排出，它包括哺乳动物细胞中没有的蛋白质，如钙依赖蛋白激酶家族(CDPKs)。我们最近发现，一种特定的弓形虫CDPK，TgCDPK3，是有效的出口、分裂、钙稳态和体内慢性感染建立所必需的，可能是通过维持弓形虫正常的生理状态来实现的。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蛋白质组学方法测量了野生型和TgCDPK3突变寄生虫的相对磷酸化位点使用量。这一分析揭示了代表106个蛋白质的156个肽以TgCDPK3依赖的方式被磷酸化，其中许多与运动性、离子稳态和新陈代谢有关。作为补充方法，我们对相互作用的蛋白质进行了基于生物素化的筛选，并鉴定了13个与TgCDPK3相关的蛋白质，其中7个在突变菌株的磷蛋白质组中也被鉴定为不那么丰富。我们推测，TgCDPK3和钙依赖的磷酸酶等酶对特定蛋白质网络的磷酸化状态的调节对于完成弓形虫的裂解周期是必不可少的。与这一想法一致的是，化学抑制TgCDPK3或磷酸酶钙调神经磷酸酶都会破坏寄生虫从宿主细胞中排出。这项提议的主要目的是描述我们所说的裂解周期的“磷酸程序”。为了做到这一点，我们将：1)通过产生和鉴定每个TgCDPK3的突变寄生虫系并确定它们的细胞定位，确定10个TgCDPK3假定底物在裂解周期中的作用。2)在已鉴定的156个亚磷酸盐网络中鉴定磷酸化事件的序列和磷酸化的化学计量。我们将使用一种高度创新的靶向蛋白质组方法来实现这一点，这种方法允许非常快速和高覆盖率的磷酸蛋白质组分析。使用不同的突变菌株，我们还将确定作为网络本身的一部分的几种蛋白质在信号级联中的相对贡献。3)通过从基因上干扰钙依赖的磷酸酶钙调神经磷酸酶并使用两种互补的方法来定义它调节的蛋白质，来确定钙依赖的磷酸酶钙调神经磷酸酶在出口过程中的功能和底物。同时，这些研究将提供对磷酸化如何调节致病寄生虫弓形虫繁殖的深入了解，这无疑将揭示寄生虫生物学中的脆弱性，可用于开发新的治疗方法。

 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.