Functional Analysis of Protein O-Glycosylation in Regulating Nuclear Growth Repressor DELLA and Plant Development in Arabidopsis

拟南芥核生长抑制因子 DELLA 蛋白 O-糖基化调节及植物发育的功能分析

• 批准号: 9899248
• 负责人: Tai-Ping Sun
• 金额: $ 31.14万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899248
• 关键词: Affect; Affinity; Algae; Animals; Antibodies; Arabidopsis; Arabidopsis Proteins; Bacteria; Binding; Biochemical Genetics; Biological Assay; Biological Process; Biology; Cell physiology; Chemicals; Complex; Cues; Data; Defect; Development; Diabetes Mellitus; Dissociation; Electron Transport; Environment; Enzymes; Eukaryota; Event; Expression Profiling; Fucose; Fucosyltransferase; Gene Expression; Genes; Genetic; Genetic Transcription; Genetic study; Genomics; Gibberellins; Growth; Growth and Development function; Human; In Vitro; Knowledge; Label; Lectin; Link; Malignant Neoplasms; Mediating; Metabolic; Methods; Modification; Monitor; Neurodegenerative Disorders; Nuclear; Nuclear Proteins; Nutrient; O-GlcNAc transferase; Organism; Orthologous Gene; Parasites; Pathway interactions; Pattern; Phenotype; Physiological; Plant Growth Regulators; Plants; Play; Post-Translational Protein Processing; Process; Protein Analysis; Protein Glycosylation; Proteins; Proteome; Proteomics; Publishing; RNA analysis; Reaction; Regulator Genes; Role; Signal Pathway; Signal Transduction; Specificity; Structure; System; Testing; Tobacco; Transcriptional Regulation; base; cell growth; genetic analysis; glycosylation; human disease; insight; knock-down; metabolomics; mutant; novel; overexpression; paralogous gene; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; plant growth/development; protein function; protein protein interaction; response; sensor; sugar; transcription factor; transcriptome; transcriptome sequencing

Project Summary Plant development requires strict coordination among complex internal signaling networks to enhance adaptation to changing environments. The conserved transcription regulators DELLA proteins play a central role in this process via direct protein-protein interactions with key transcription factors. Recent studies using genetic and physiological analyses together with chemical biology methods indicate that DELLA's binding affinity to interacting proteins are oppositely regulated by two types of O-linked glycosylation on specific Ser/Thr residues: O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-fucosylation (O-Fuc). These two distinct O-glycosyl modifications on DELLA are catalyzed by two paralogs in Arabidopsis: SECRET AGENT (SEC), an O-GlcNAc transferase (OGT) that reduces DELLA activity, and SPINDLY (SPY), a novel protein O-fucosyltransferase (POFUT) that enhances DELLA activity. These studies uncovered direct roles of OGT (SEC) and POFUT (SPY) in fine-tuning plant development by modulating DELLA interactions with key regulators in multiple signaling pathways. OGT-mediated protein O-GlcNAcylation has been studied extensively in animals, and is known to play a key role in regulating a plethora of intracellular signaling events in response to nutrient status. In contrast, the physiological functions of OGT in plants are largely unknown. Moreover, SPY is the first POFUT identified for O-fucosylation of nuclear proteins, uncovering a novel mechanism for transcriptional regulation. The dynamic interplay between O-GlcNAc/O-Fuc modifications in regulating the nuclear growth repressor DELLA activity may provide a new paradigm in linking metabolic status to gene expression and cell growth in response to internal and external cues. This hypothesis will be tested using targeted metabolomics and chemical biology approaches (Specific Aim 1). In addition, structure analysis of SPY and SPY/substrate complexes will identify key residues that contribute to the substrate and enzymatic specificity of SPY. The interplay between O-GlcNAc and O-Fuc is likely to modulate diverse cellular activities beyond DELLA function. The pleiotropic phenotypes of spy and sec mutants, recently published Arabidopsis O-GlcNAc proteome and preliminary results in this lab suggest that many Arabidopsis proteins involved in transcriptional control are common targets of SEC and SPY. The global roles of SPY and SEC in plant development will be elucidated by genetic studies using inducible knockdown/overexpression SPY and SEC lines in conjunction with genomic and proteomic approaches (Specific Aim 2). This study will have broader implications. SPY orthologs, although absent in animals, are highly conserved in diverse organisms, including plants, bacteria, and parasitic protists, suggesting that intracellular O-fucosylation regulates a wide range of biological processes in diverse organisms.

项目摘要 植物的发育需要复杂的内部信号网络之间的严格协调，以增强 适应不断变化的环境。保守的转录调节因子DELLA蛋白在转录调控中起着重要作用。 通过与关键转录因子的直接蛋白质-蛋白质相互作用在这一过程中发挥作用。最近的研究使用 遗传和生理分析以及化学生物学方法表明DELLA的结合 与相互作用蛋白质的亲和力由两种类型的O-连接的糖基化在特定的 Ser/Thr残基：O-连接的N-乙酰葡糖胺（O-GlcNAc）修饰和O-岩藻糖基化（O-Fuc）。这些 DELLA上的两种不同的O-糖基修饰由拟南芥中的两种旁系同源物催化：SECRET AGENT（SEC），一种降低DELLA活性的O-GlcNAc转移酶（OGT），和SPINDLY（SPY），一种新的 蛋白质O-岩藻糖基转移酶（POFUT），增强DELLA活性。这些研究揭示了 OGT（SEC）和POFUT（SPY）通过调节DELLA与关键基因的相互作用来微调植物发育 调节多种信号通路。研究了OGT介导的蛋白质O-GlcNAc化 在动物中广泛存在，并且已知在调节过多的细胞内信号事件中起关键作用 以响应营养状况。相反，OGT在植物中的生理功能在很大程度上是未知的。 此外，SPY是第一个鉴定为核蛋白O-岩藻糖基化的POFUT，揭示了一种新的 转录调控机制。O-GlcNAc/O-Fuc修饰之间的动态相互作用 调节细胞核生长抑制因子DELLA的活性可能提供一种新的模式， 状态对基因表达和细胞生长的响应内部和外部线索。这一假设将是 使用靶向代谢组学和化学生物学方法进行测试（具体目标1）。此外，结构 SPY和SPY/底物复合物的分析将鉴定对底物有贡献的关键残基， SPY的酶特异性。O-GlcNAc和O-Fuc之间的相互作用可能会调节多种 除了DELLA功能之外的细胞活动。spy和sec突变体的多效性表型，最近 发表的拟南芥O-GlcNAc蛋白质组和本实验室的初步结果表明，许多拟南芥 参与转录控制的蛋白质是SEC和SPY的共同靶标。SPY的全球角色， SEC在植物发育中的作用将通过使用诱导性敲低/过表达SPY的遗传研究来阐明 和SEC系结合基因组和蛋白质组学方法（具体目标2）。这项研究将有 更广泛的影响。SPY直向同源物虽然在动物中不存在，但在不同生物体中高度保守， 包括植物，细菌和寄生原生生物，表明细胞内O-岩藻糖基化调节广泛的 在不同的生物体中的一系列生物过程。