Phosphoproteome of Toxoplasma

描述(申请人提供)：Apicomplexan寄生虫导致人类许多疾病，包括疟疾和弓形虫病。它们通过入侵、复制和退出人类细胞来做到这一点。近年来，关于执行这些过程的分子已经了解了很多，但它们的行动是如何协调的，信号是如何接收来启动每个过程的，在很大程度上仍然是个谜。从其他生物体的研究中得知，蛋白质的磷酸化是调节其作用的最重要的手段之一。最近，质谱学技术已经出现，可以大规模鉴定几乎整个这样的修饰集；这个数据集被称为“磷蛋白质组”。我们提出的工作的目标是确定在细胞内和细胞外条件下，弓形虫急性阶段发生的磷酸蛋白质组变化，所谓的速殖子。为了使我们能够确定哪些变化是这些过程的关键，我们将使用特定的药物来扰乱钙依赖事件，这些事件是已知的入侵和排出的关键。我们还将战略性地利用现有的弓形虫突变株，这些突变株在与入侵/出口相关的信号转导方面存在缺陷。最后，我们将利用我们在质谱学方面的专业知识，为那些可用数量太少而无法进行常规分析的样本创新磷酸蛋白质组学分析方法。

DESCRIPTION (provided by applicant): Apicomplexan parasites cause many diseases in humans, including malaria and toxoplasmosis. They do this by invading, replicating and exiting human cells. In recent years, much has been learned about the molecules that carry out these processes but how their actions are coordinated and how signals are received to initiate each process have remained largely mysterious. From studies in other organisms, it is known that phosphorylation of proteins is one of the most important means by which their action is regulated. Recently, mass spectrometric techniques have emerged that allow the large-scale identification of the nearly entire set of such modifications; this dataset is known as the "phosphoproteome". The goal of our proposed work is to determine the phosphoproteomic changes that occur in the acute stage of Toxoplasma parasites, the so-called tachyzoites, under intracellular vs. egressing conditions. To enable us to determine which changes are key to these processes, we will use specific drugs that disrupt calcium-dependent events that are known to be key to invasion and egress. We will also make strategic use of existing mutant lines of Toxoplasma that are defective in signaling related to invasion/egress. Finally, we will take advantage of our expertise in mass spectrometry to innovate new methods for phosphoproteomic analyses of samples where the amount available is too small for conventional analyses.