Dissecting the roles of protein O-GlcNAcylation in Toxoplasma gondii

描述（由申请人提供）：弓形虫是顶复门中的专性细胞内寄生虫。这种寄生虫会导致艾滋病患者和免疫功能低下的人患上危及生命的疾病，并且当妇女在怀孕期间感染时，会导致出生缺陷或流产。弓形虫在人类宿主中发病和持续存在的一个重要过程是快速分裂的速殖子和潜伏的缓殖子之间的相互转化。尽管其具有病理意义，但对该过程背后的信号分子和机制知之甚少。 最近，在弓形虫中发现了普遍存​​在的 O-GlcNAc 酰化和 O-GlcNAc 转移酶（OGT）（催化这一过程的酶）。 OGT 将单个 ¿-N-乙酰氨基葡萄糖添加到细胞内蛋白质的丝氨酸或苏氨酸残基上。因为在哺乳动物中，O-GlcNAc 是细胞内信号传导、转录调节和蛋白质运输的介质，以响应细胞营养或应激，我们假设 O-GlcNAc 可能通过动态修饰细胞内蛋白质，在弓形虫生命周期在速殖子和缓殖子之间的转变中发挥积极作用。为了检验这一假设，我们将使用化学酶法来鉴定弓形虫速殖子中的 O-GlcNAc 修饰蛋白（目标 1）。接下来，我们将应用该方法对弓形虫速殖子-缓殖子转变过程中蛋白质 O-GlcNAc 状态的变化进行比较分析。我们将把研究重点放在参与这一过程的基因调节蛋白上。一旦确定，我们将 描述 O-GlcNAc 在介导这些修饰蛋白在此生命周期转变中的生物学功能中的作用（目标 2）。最后，我们将描述全球变化的特征 OGT 介导的蛋白质 O-GlcNAc 酰化影响弓形虫的生命周期转变（目标 3）。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa. This parasite causes life-threatening diseases in AIDS patients and immune-compromised individuals, and birth defects or abortions when women are infected during pregnancy. An essential process for pathogenesis and persistence of T. gondii in human hosts is the interconversion between the rapidly dividing tachyzoites and latent bradyzoites. Despite its pathological significance, the signaling molecules and the mechanisms underlying this process are poorly understood. Recently, ubiquitous O-GlcNAcylation and O-GlcNAc transferase (OGT), the enzyme that catalyzes this process, have been identified in T. gondii. OGT adds a single ¿-N-acetylglucosamine to serine or threonine residues of intracellular proteins. Because in mammalian organisms, O-GlcNAc is a mediator of intracellular signaling, transcription regulation, and protein trafficking in response to cellular nutrients or stress, we hypothesize that O-GlcNAc may serve as an active player in T. gondii's life cycle transition between tachyzoites and bradyzoites by dynamically modifying intracellular proteins. To test this hypothesis, we will use a chemoenzymatic approach to identify O-GlcNAc-modified proteins in T. gondii tachyzoites (Aim 1). Next, we will apply this approach for comparative analysis of changes in protein O-GlcNAc status in T. gondii tachyzoite-bradyzoites transition. We will focus our studies on gene-regulatory proteins that are involved in this process. Once identified, we will characterize the role of O-GlcNAc in mediating the biological functions of these modified proteins in this life-cycle transition (Aim 2). Finally, we will characterize how changes in global protein O-GlcNAcylation mediated by OGT influence the life-cycle transition of T. gondii (Aim 3).