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

• 批准号: 10541113
• 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
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10541113
• 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; Eye diseases; Felis catus; Fetus; Fucose; Fucosyltransferase; Future; Genes; Genetic Transcription; Green Fluorescent Proteins; Growth; Guanosine Diphosphate Fucose; Guanosine Triphosphate Phosphohydrolases; Histones; Homologous Gene; Human; Hydrolysis; Hydroxyproline; Infection; Knock-out; Knowledge; Label; Lectin; Life Cycle Stages; Lung infections; Mass Spectrum Analysis; Membrane; Messenger RNA; Methods; Microscopy; Modification; Mus; N-terminal; 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; Toxoplasma; Toxoplasma gondii; Transferase; Virulence; Work; X-linked intellectual disability; experimental study; fitness; gibberellic acid; glycosylation; glycosyltransferase; human model; human pathogen; immunosuppressed; inorganic phosphate; molecular phenotype; mouse model; nervous system disorder; novel; overexpression; p19(SKP1) Protein; paralogous gene; proteostasis; public health relevance; response; sugar; transcription factor; transcriptome sequencing; ubiquitin-protein ligase

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.

项目摘要/摘要 该提案探讨了核O-凝结物作为弓形虫中新型调节机制的作用 Gondii，严重的神经和眼疾病的原因。约翰·萨缪尔森（John Samuelson）实验室使用了抗凝血藻 Aurantia凝集素（AAL）表明，T. gondii的岩藻糖基化的蛋白质形成点状组件，紧密结合 与核孔复合体。 O羟化蛋白的组件让人联想到Cajal，PML和 宿主核中的组蛋白基因座体，该基因核未被O-谷物修饰。 AAL-ENCHMENT和质量 光谱法显示O-谷物附着于Ser和THR，并在数十个本质上无序的区域 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 Lab确定了gondii O-转移酶（TGSPY），其中包含11个N末端四肽重复序列 （TPRS）和C末端GT41糖基转移酶结构域。重组TGSPY在细胞质中制成 细菌水解GDP-葡萄糖和O藻糖基酸盐本身，多级别剂/GST融合以及裂解物中的蛋白质 Spyko细胞。 TGSPY不是必需的，但是Spyko的增长较慢，而转染者过表达 TGSPY生长更快。融合与多聚体融合的绿色荧光蛋白是O型糖基化的，并积聚在AAL- 在父菌株中标记为组件，但在spyko中降解。 TGSPY是植物的同源物，拟南芥中的gibberellic Acid信号的负调节剂，IS 也是旁系同源物宿主O-GLCNAC转移酶（OGT）。 TG中不存在的OGT可以固有地修饰 数百种蛋白质区域的无序区域是应激性和养分信号传导的重要因素。 根据OGT提供的先例，我们假设TGSPY修改了蛋白质并影响其活性 在正常生长条件下，应对压力。此外，TGSPY唯一的修改 O羟化蛋白可在NPC附近定位，并在其中保护它们免受降解。 萨缪尔森（Samuelson）和西部实验室（West Labs）将共同努力，以剖析O-upose在T. Gondii中的工作方式。在目标1中我们 将评估TPRS和GT41糖基转移酶对O型糖基化和寄生虫生长的重要性。 在AIM 2中，我们将确定五种蛋白质的稳定性，定位和功能，这些蛋白是O型核苷酸化的，并且 响应不同的间谍表达而有助于TG适应性。我们将进行互动研究以解决 蛋白质包含在O型糖基化蛋白质和脉冲式标记的组件中的机制，时间 - 裂开显微镜和照片漂白以确定组件的稳定性。在AIM 3中，我们将比较 父母菌株的培养，小鼠和猫的生长，间谍敲除和过表达TGSPY的生物。 此外，我们将对三组生长为速剂或 在文化中转变为牛皮二氮。