The Biochemistry and Cell Biology of the Spindly O-fucosyltransferase of Toxoplasma

项目概要/摘要 该建议探讨了核O-岩藻糖在弓形虫中作为一种新的调节机制的作用 弓形虫，导致严重的神经和眼部疾病。约翰·萨缪尔森实验室用抗岩藻糖粉糊 橙黄凝集素（AAL），以显示岩藻糖基化的T.弓形虫形成紧密结合的点状集合体 与核孔复合体结合。O-岩藻糖基化蛋白的组装让人想起Cajal、PML和 宿主细胞核中的组蛋白基因座体，其不被O-岩藻糖修饰。AAL富集和质量 光谱法显示，O-岩藻糖在数十个固有无序区域中连接到Ser和Thr上 这些蛋白质似乎参与转录、mRNA加工和运输以及信号传导。克里斯 西部实验室确认了T弓形虫O-转移酶（TgSpy），其含有11个N-末端三肽重复 在一个实施方案中，所述糖基转移酶包含TPRs和C-末端GT 41糖基转移酶结构域。重组TgSpy在细胞质中制备， 细菌水解GDP-岩藻糖和O-岩藻糖基化物本身，一种聚丝氨酸/GST融合物，以及来自 SpyKO细胞。TgSpy不是必需的，但spyKO生长较慢，而过表达的转染子 TgSpy增长更快。与polySer融合的绿色荧光蛋白被O-岩藻糖基化并在AAL-中积累 标记的组装体在亲本菌株中，但在spyKO中降解。 TgSpy是植物Spindly的同源物，Spindly是拟南芥中赤霉酸信号传导的负调节因子， 也是宿主O-GlcNAc转移酶（OGT）的一部分。在Tg中不存在的OGT本质上改变了 它是数百种蛋白质的无序区域，是抗应激和营养信号传导的重要因素。 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。此外，TgSpy的修改独特地导致 0-岩藻糖基化蛋白定位在NPC附近，在那里它们被保护免于降解。 萨缪尔森和韦斯特实验室将共同研究O-岩藻糖在T。刚地。在目标1中， 将评估TPR和GT 41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，这五种蛋白质是O-岩藻糖基化的， 有助于Tg健身，以应对不同的间谍表达。我们将进行相互作用组研究， O-岩藻糖基化蛋白质组装体中蛋白质内含物的机制和脉冲追踪标记，时间- lapse显微镜和光漂白来确定组装体的稳定性。在目标3中，我们将比较 亲本菌株、spy敲除菌株和过表达TgSpy的生物体在培养物、小鼠和猫中的生长。 此外，我们将对三组原生生物进行RNA-seq和SILAC，这些原生生物生长为速殖子， 在培养中转化为缓殖子。

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.