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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要植物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 蛋白发挥着核心作用通过与关键转录因子的直接蛋白质-蛋白质相互作用，在此过程中发挥作用。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的亲和力受到特定的两种类型的 O-连接糖基化的相反调节 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 上的两个不同的 O-糖基修饰由拟南芥中的两个旁系同源物催化：秘密 AGENT (SEC)，一种可降低 DELLA 活性的 O-GlcNAc 转移酶 (OGT)，以及 SPINDLY (SPY)，一种新型蛋白质 O-岩藻糖基转移酶 (POFUT) 可增强 DELLA 活性。这些研究揭示了 OGT (SEC) 和 POFUT (SPY) 通过调节 DELLA 与关键蛋白的相互作用来微调植物发育多个信号通路中的调节因子。 OGT 介导的蛋白质 O-GlcNAc 酰化已被研究广泛存在于动物体内，已知在调节大量细胞内信号传导事件中发挥关键作用以响应营养状况。相比之下，OGT 在植物中的生理功能很大程度上是未知的。此外，SPY 是第一个被鉴定用于核蛋白 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核生长抑制因子 DELLA 活性可能提供联系代谢的新范例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进行测试（具体目标 1）。另外，结构 SPY 和 SPY/底物复合物的分析将识别有助于底物和 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超出 DELLA 功能的细胞活动。最近，spy 和 sec 突变体的多效性表型已发表的拟南芥 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 的共同目标。 SPY 的全球角色和植物发育中的 SEC 将通过使用诱导型敲低/过度表达 SPY 的遗传研究来阐明和 SEC 系与基因组和蛋白质组学方法相结合（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，但在多种生物体中高度保守，包括植物、细菌和寄生原生生物，表明细胞内 O-岩藻糖基化广泛调节不同生物体中的一系列生物过程。