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

弓形虫 SpindlyO-岩藻糖基转移酶的生物化学和细胞生物学

基本信息

批准号：
10300056
负责人：
John C. Samuelson
金额：
$ 51.63万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10300056
关键词：
Acanthamoeba Adaptor Signaling Protein Address Adult Affect Aleuria aurantia lectin Amino Acids Amino Sugars Antibodies Arabidopsis Bacteria Biochemistry Biology Blindness Brain Burkholderia C-terminal Catalytic Domain Category B pathogen Cell Nucleus Cells Cellular biology Chemistry Chimeric Proteins Complement Cryptosporidium Cyst Cytoplasm Defect Diarrhea Dictyostelium Differentiation and Growth Disease Enzymes Felis catus Fetus Fucose Fucosyltransferase Future Genes Genetic Transcription Green Fluorescent Proteins Growth Guanosine Diphosphate Fucose Guanosine Triphosphate Phosphohydrolases Histones Homologous Gene Human Hydroxyproline Infection Knock-out Knowledge Label Lectin Life Cycle Stages Lung infections Mass Spectrum Analysis Membrane Messenger RNA Methods Microscopy Modification Mus N-terminal Neurologic Nuclear Nuclear Pore Complex Nuclear Pore Complex Proteins Nutrient O-GlcNAc transferase Organism Oxygen Parasites Parents Persons Phase Transition Photobleaching Physiologic pulse Plants Point Mutation Polymerase Post-Translational Protein Processing Protein Region Proteins Radiolabeled Reaction Recombinant Proteins Recombinants Reporter Research Personnel Resistance Role Signal Transduction Signaling Protein Site Stress Surveys System Testing Time Toxoplasma Toxoplasma gondii Transferase Virulence Work X-linked intellectual disability base experimental study fitness gibberellic acid glycosylation glycosyltransferase human model human pathogen immunosuppressed inorganic phosphate molecular phenotype mouse model novel overexpression p19(SKP1) Protein paralogous gene proteostasis public health relevance response sugar transcription factor transcriptome sequencing ubiquitin-protein ligase

项目摘要

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.

项目概要/摘要该提案探讨了核 O-岩藻糖作为弓形虫新型调节机制的作用弓形虫，导致严重的神经系统和眼部疾病。约翰·萨缪尔森实验室使用抗岩藻糖 Aleuria aurantia 凝集素 (AAL) 显示弓形虫的岩藻糖基化蛋白形成紧密关联的点状组装体与核孔复合体。 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位点体，不被 O-岩藻糖修饰。 AAL-富集和质量光谱分析显示，O-岩藻糖在数十个本质无序区域中附着在 Ser 和 Thr 上。蛋白质，似乎参与转录、mRNA 加工和运输以及信号传导。克里斯 West 实验室鉴定出弓形虫 O 转移酶 (TgSpy)，其中包含 11 个 N 末端四肽重复序列 (TPR) 和 C 端 GT41 糖基转移酶结构域。在细胞质中制备的重组 TgSpy 细菌水解 GDP-岩藻糖和 O-岩藻糖基化物本身、多聚 Ser/GST 融合物以及裂解物中的蛋白质间谍KO细胞。 TgSpy不是必需的，但spyKO生长得更慢，而过度表达的转染子 TgSpy 成长得更快。与多聚丝氨酸融合的绿色荧光蛋白被 O-岩藻糖基化并在 AAL- 中积累亲本菌株中标记的组装体但在spyKO中被降解。 TgSpy 是植物 Spindly 的同源物，是拟南芥中赤霉酸信号传导的负调节因子，并且是也是宿主 O-GlcNAc 转移酶 (OGT) 的旁系同源物。 Tg 中不存在的 OGT 本质上改变了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 的修改会导致 O-岩藻糖基化蛋白质定位在 NPC 附近，在那里它们受到保护免遭降解。 Samuelson 和 West 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，这些蛋白质是 O-岩藻糖基化和有助于 Tg 适应性，以响应不同的 Spy 表达。我们将进行相互作用组研究来解决 O-岩藻糖基化蛋白质组装中的蛋白质包含机制和脉冲追踪标记、时间- 延时显微镜和光漂白以确定组件的稳定性。在目标 3 中，我们将比较亲本品系、spy 敲除和过度表达 TgSpy 的生物体在培养物、小鼠和猫中的生长。此外，我们将对三组作为速殖子或速殖子生长的原生生物进行RNA-seq和SILAC 在培养物中转化为缓殖子。