Phosphoproteome of Toxoplasma

描述（由申请人提供）：顶复门寄生虫引起人类多种疾病，包括疟疾和弓形虫病。它们通过入侵、复制和退出人体细胞来做到这一点。近年来，人们对执行这些过程的分子有了很多了解，但它们的行为是如何协调的，以及如何接收信号以启动每个过程，在很大程度上仍然是个谜。从其他生物的研究中，已知蛋白质的磷酸化是调节其作用的最重要手段之一。最近，质谱技术已经出现，允许大规模识别的几乎整个集这样的修改，这个数据集被称为“磷酸化蛋白质组”。我们提出的工作的目标是确定磷酸化蛋白质组的变化，发生在急性期的弓形虫寄生虫，所谓的速殖子，在细胞内与出口条件下。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破坏钙依赖性事件，这些事件已知是入侵和出口的关键。我们还将战略性地利用现有的弓形虫突变株系，这些突变株系在与入侵/外出相关的信号传导中存在缺陷。最后，我们将利用我们在质谱方面的专业知识，创新用于磷酸化蛋白质组学分析样品的新方法，其中可用的量对于常规分析来说太小。

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.