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 植物开发需要在复杂的内部信号网络之间进行严格的协调以增强 适应不断变化的环境。保守的转录调节剂DELLA蛋白发挥了中心 通过直接蛋白质 - 蛋白质与关键转录因子相互作用在此过程中的作用。最近使用的研究 遗传和生理分析与化学生物学方法一起表明Della的结合 对相互作用蛋白的亲和力由两种类型的O连接糖基化的特异性调节 Ser/THR残基：O连锁的N-乙酰葡萄糖（O-GLCNAC）修饰和O羟基化（O-FUC）。这些 拟南芥中的两个旁系同源物催化了两种不同的O-糖基修饰：秘密 代理（SEC），一种O-GlCNAC转移酶（OGT），可降低DELLA活性，而Spindly（间谍），一种新颖的 蛋白O-核糖基转移酶（POFUT），可增强DELLA活性。这些研究发现了 OGT（SEC）和POFUT（间谍）在微调植物开发中通过调节Della相互作用与钥匙 多个信号通路中的调节器。已经研究了OGT介导的蛋白O-Glcnacylation 在动物中广泛，众所周知，在调节大量细胞内信号事件中起着关键作用 响应营养状况。相反，植物中OGT的生理功能在很大程度上未知。 此外，间谍是鉴定出用于核蛋白的O型核糖基化的第一个pofut，发现了一种新颖的 转录调控机制。 O-GLCNAC/O-FUC修改之间的动态相互作用 调节核生长阻遏物DELLA活性可能会提供新的范式 响应内部和外部线索的基因表达和细胞生长状态。这个假设将是 使用靶向代谢组学和化学生物学方法测试（特定目标1）。另外，结构 间谍和间谍/底物复合物的分析将确定有助于底物的关键残基和 间谍的酶特异性。 O-GLCNAC和O-FUC之间的相互作用可能会调节多样化 除了DELLA功能之外的细胞活动。间谍和SEC突变体的多效性表型，最近 该实验室中已发表的拟南芥O-GlcNAC蛋白质组和初步结果表明许多拟南芥 参与转录控制的蛋白质是SEC和间谍的常见靶标。间谍和 使用诱导的敲低/过表达间谍，遗传研究将阐明植物发育中的SEC SEC线与基因组和蛋白质组学方法结合使用（特定目标2）。这项研究将有 更广泛的含义。间谍直系同源物虽然在动物中不存在，但在多种生物中是高度保守的，但是 包括植物，细菌和寄生虫生物，表明细胞内O-凝血素化调节宽 各种生物体的生物过程范围。