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

项目摘要 工厂的发展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 通过蛋白质-蛋白质与关键转录因子的直接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 与相互作用蛋白的亲和力受两种类型的O-连接糖基化的相反调节 丝氨酸/苏氨酸残基：O-连接N-乙酰氨基葡萄糖(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Paralog催化：秘密 试剂(SEC)，一种降低Della活性的O-GlcNAc转移酶(OGT)，以及一种新型的纺锤状(SPY) 增强Della活性的蛋白O-岩藻糖基转移酶(POFUT)。这些研究揭示了 OGT(SEC)和POFUT(SPY)通过调节Della与KEY的相互作用来微调植物发育 多条信号通路中的调节因子。OGT介导的蛋白质O-GlcN酰化反应的研究 广泛存在于动物体内，已知在调节过多的细胞内信号事件中起关键作用 对营养状况的反应。相比之下，OGT在植物中的生理功能在很大程度上是未知的。 此外，SPY是第一个被鉴定为核蛋白O-岩藻糖基化的POFUT，发现了一种新的 转录调控机制。O-GlcNAc/O-Fuc修饰间的动态相互作用 调节核生长抑制因子della的活性可能会提供一种新的研究范式 对基因表达和细胞生长的状态对内部和外部提示的响应。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 对间谍和间谍/底物复合体的分析将确定有助于底物和底物的关键残基 Spy的酶特异性。O-GlcNAc和O-Fuc之间的相互作用可能调节不同的 Della功能之外的细胞活动。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 参与转录调控的蛋白质是SEC和SPY的共同靶标。间谍和间谍的全球角色 植物发育中的SEC将通过使用可诱导的敲除/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方法(特定目标2)。这项研究将会有 更广泛的影响。Spy同源基因虽然在动物中不存在，但在不同的生物体中高度保守， 包括植物、细菌和寄生原生生物，这表明细胞内的O-岩藻糖化调节广泛的 不同生物体的一系列生物过程。

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.