The Biochemistry and Cell Biology of the SpindlyO-fucosyltransferase of Toxoplasma

项目摘要/摘要 这项建议探讨了核O-岩藻糖作为弓形虫新的调节机制的作用。 弓形虫病，导致严重的神经和眼部疾病。John Samuelson实验室使用了抗岩藻糖阿列乌里亚 AAL表明弓形虫岩藻糖化蛋白形成紧密结合的点状集合体 核孔复合体。O-岩藻糖化蛋白的组装使人联想到Cajal、PML和 组蛋白在宿主核内，不被O-岩藻糖修饰。AAL的浓缩和质量 光谱分析显示O-岩藻糖结合在丝氨酸和苏氨酸上，在数十个 蛋白质，它们似乎参与转录、信使核糖核酸的处理和运输，以及信号传递。《克里斯》 West实验室鉴定了弓形虫O-转移酶(TgSpy)，它含有11个N端四肽重复序列 (TPR)和一个C端的GT41糖基转移酶结构域。重组人TgSpy的细胞质制备 细菌本身可以降解GDP-岩藻糖和O-岩藻糖酸盐，形成PolySer/GST融合，并在裂解产物中产生蛋白质 SpyKO细胞。TgSpy不是必需的，但spyKO生长较慢，而过度表达的转染体 TgSpy成长得更快。与PolySer融合的绿色荧光蛋白被O-岩藻糖基化并聚集在AAL- 在亲本菌株中标记的组件，但在spyKO中被降解。 TgSpy是拟南芥中赤霉酸信号的负调控因子，是植物Spinly的同系物，是 与宿主O-GlcNAc转移酶(OGT)类似。在TG中没有的OGT本质上是修饰的 它是数百种蛋白质的无序区域，是抗逆性和营养信号的重要因素。 根据OGT提供的先例，我们假设TgSpy修饰蛋白质并影响其活性 在正常生长条件下和对压力的反应中。此外，TgSpy的修改唯一导致 O-岩藻糖化蛋白定位在鼻咽癌附近，在那里它们受到保护，不会被降解。 萨缪尔森实验室和韦斯特实验室将共同研究O-岩藻糖在弓形虫中的作用。在目标1中，我们 将评估TPR和GT41糖基转移酶对O-岩藻糖基化和寄生虫生长的重要性。 在目标2中，我们将确定五种蛋白质的稳定性、定位和功能，它们是O-岩藻糖化的和 有助于TG健身，回应不同的Spy表情。我们将进行互动组研究，以解决 研究了O-岩藻糖化蛋白组装过程中的蛋白质包合机制和脉冲追逐标记、时间-时间标记等。 失光显微镜和光漂白以确定组件的稳定性。在目标3中，我们将比较 在培养、小鼠和猫的亲本菌株、间谍基因敲除和过度表达TgSpy的生物中生长。 此外，我们将对以速殖子或SILAC形式生长的三组原生生物进行RNA-SEQ和SILAC 在培养过程中转变为缓殖子。

Project summary/Abstract This proposal explores the role of nuclear O-fucose as a novel regulatory mechanism in Toxoplasma gondii, cause of severe neurological and ocular diseases. The John Samuelson lab used the anti-fucose Aleuria aurantia lectin (AAL) to show that fucosylated proteins of T. gondii form punctate assemblies in close association with the nuclear pore complex. The assemblies of O-fucosylated proteins are reminiscent of Cajal, PML, and histone locus bodies in the host nucleus, which are not modified by O-fucose. AAL-enrichment and mass spectrometry showed O-fucose is attached to Ser and Thr in intrinsically disordered regions of dozens of proteins, which appear to be involved in transcription, mRNA processing and transport, and signaling. The Chris West lab identified the T. gondii O-transferase (TgSpy), which contains 11 N-terminal tetratricopeptide repeats (TPRs) and a C-terminal GT41 glycosyltransferase domain. Recombinant TgSpy made in the cytoplasm of bacteria hydrolyses GDP-fucose and O-fucosylates itself, a polySer/GST fusion, and proteins in lysates from spyKO cells. TgSpy is not essential, but the spyKO grows more slowly, while transfectants that overexpress TgSpy grow faster. Green fluorescent protein fused with polySer is O-fucosylated and accumulates in the AAL- labeled assemblies in the parent strain but is degraded in the spyKO. TgSpy is a homolog of plant Spindly, a negative regulator of gibberellic acid signaling in Arabidopsis, and is also a paralog the host O-GlcNAc transferase (OGT). The OGT, which is absent in Tg, modifies intrinsically disordered regions of hundreds of proteins and is an important factor in stress resistance and nutrient signaling. According to precedent provided by OGT, we hypothesize that TgSpy modifies proteins and affects their activity under normal growth conditions and in response to stress. In addition, modification by TgSpy uniquely causes O-fucosylated proteins to localize near the NPC, where they are protected from degradation. The Samuelson and West labs will work together to dissect how O-fucose works in T. gondii. In Aim 1 we will evaluate the importance of the TPRs and GT41 glycosyltransferase for O-fucosylation and parasite growth. In Aim 2 we will determine the stability, localization, and function of five proteins, which are O-fucosylated and contribute to Tg fitness, in response to varied Spy expression. We will perform interactome studies to address the mechanism of protein inclusion in assemblies of O-fucosylated proteins and pulse-chase labeling, time- lapse microscopy, and photo-bleaching to determine the stability of assemblies. In Aim 3 we will compare the growth in culture, mice, and cats of the parental strain, the spy knockout, and organisms overexpressing TgSpy. In addition, we will perform RNA-seq and SILAC on the three sets of protists growing as tachyzoites or converting to bradyzoites in culture.