Dissecting the calcium dependent phosphorylation network of Toxoplasma gondii

 描述（由申请人提供）：顶复门的一些细胞内寄生虫，如恶性疟原虫和刚地弓形虫，是影响人类的最重要的传染源之一。由于猖獗的耐药性、毒性和缺乏针对某些阶段的活性，迫切需要新的抗顶复门药物。这些寄生虫中的钙信号传导作为潜在的药物靶点值得特别关注，因为它驱动许多基本事件，如运动性、侵入和外出，并且它包括哺乳动物细胞中未发现的蛋白质，如钙依赖性蛋白激酶（CDPK）家族。我们最近发现，一个特定的弓形虫CDPK，TgCDPK3，需要有效的出口，分裂，钙稳态和建立一个慢性感染体内，大概是通过维持正常的生理状态，其中寄生虫的功能。为了理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。该分析揭示了156种肽，代表以TgCDPK3依赖性方式磷酸化的106种蛋白质，其中许多与运动、离子稳态和代谢有关。作为一种补充方法，我们进行了基于生物素化的相互作用蛋白质的筛选，并确定了13个推定的TgCDPK3相关蛋白，其中7个也被确定为在突变株的磷酸化蛋白质组中丰度较低。我们推测，通过酶如TgCDPK3和钙依赖性磷酸酶调节特定蛋白质网络的磷酸化状态对于弓形虫的裂解周期的完成是必不可少的。与这一想法一致，TgCDPK3或磷酸酶钙调磷酸酶的化学抑制破坏寄生虫从宿主细胞的退出。这个建议的主要目标是表征我们所说的裂解循环的“磷酸化程序”。为此，我们将：1）通过产生和表征每种的突变寄生虫系并确定其细胞定位，确定十种TgCDPK3推定底物在裂解周期中的作用。2)在156个磷酸化位点的识别网络中识别磷酸化事件的顺序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学方法来实现这一目标，该方法允许非常快速和高覆盖率的磷酸化蛋白质组分析。使用各种突变株，我们还将确定几种蛋白质的相对贡献，这些蛋白质是网络本身对信号级联的一部分。3)确定功能和底物的钙依赖性磷酸酶钙调磷酸酶在出口过程中，通过遗传破坏它，并使用两个互补的方法来定义它调节的蛋白质。同时，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。

 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.