Structural and Functional Analysis of Nucleocytoplasmic Protein O-Glycosyltransferases in Plants

项目总结/摘要 O-连接的蛋白糖基化在植物发育中响应于糖基化而协调不同的信号传导事件。 不断变化的环境我们发现了第一个核质O-岩藻糖基转移酶，SPINDLY（SPY） 在拟南芥中，其与其旁腺分泌剂（SEC）一起，是一种O-GlcNAc转移酶（OGT）， 保守地修饰转录调节因子的保守DELLA家族，如RGA，以控制它们的能力， 来调节多种信号通路。SPY催化的RGA的O-岩藻糖基化增强了RGA相互作用 与关键转录因子抑制植物生长，而SEC介导的O-GlcNAc化减弱RGA 促进植物生长发育。尽管它们的功能意义，这两个同源 糖基转移酶，SPY和SEC，已经进化出不同的底物特异性，以及这两种类型的糖基转移酶是如何被激活的。 糖基化、通过SPY的O-岩藻糖基化和通过SEC的O-GlcNAc化，调节RGA构象以影响 植物在分子水平上的发育仍然是未知的。我们建议阐明分子细节 SPY、SEC及其底物和产物复合物，以及SPY、SEC的不同结构和功能 RGA O-岩藻糖基化和O-GlcNAc酰化的结果。最近的蛋白质组分析揭示了数百个 RGA以外的SPY和SEC底物的合成，强调了蛋白质糖基化在调节中的广泛意义 细胞功能。为了克服植物中spy和sec突变所带来的潜在多效性效应， 并破译特定靶蛋白的O-岩藻糖基化和O-GlcNAc酰化的功能结果， 我们将开发融合纳米抗体的SPY和SEC变体，用于精确的靶向修饰。总的来说，我们的研究 将填补关键的知识空白，蛋白质糖基化的结构和功能的后果， 植物由于SPY样基因在包括植物、细菌和寄生原生生物在内的多种生物中是保守的， 由于SEC样（OGT）基因广泛分布于植物和动物中，因此对这些基因的分子理解 酶及其独特的结构和功能结果将对植物生物学产生深远的影响， 农业、人类健康和疾病。

Project Summary/Abstract O-linked protein glycosylation orchestrates diverse signaling events in plant development in response to the changing environment. We have discovered the first nucleocytoplasmic O-fucosyltransferase, SPINDLY (SPY) in Arabidopsis, which together with its paralog SECRET AGENT (SEC), an O-GlcNAc transferase (OGT), post- translationally modify the conserved DELLA family of transcription regulators, such as RGA, to control their ability to modulate multiple signaling pathways. SPY-catalyzed O-fucosylation of RGA enhances the RGA interaction with key transcription factors to suppress plant growth, whereas SEC-mediated O-GlcNAcylation weakens RGA activity and promotes plant development. Despite their functional significance, how these two homologous glycosyltransferases, SPY and SEC, have evolved distinct substrate specificities and how these two types of glycosylation, O-fucosylation by SPY and O-GlcNAcylation by SEC, regulate the RGA conformation to influence plant development at the molecular level have remained unknown. We propose to elucidate the molecular details of SPY, SEC, and their substrate and product complexes, as well as the distinct structural and functional consequences of RGA O-fucosylation and O-GlcNAcylation. Recent proteomic analysis has unveiled hundreds of SPY and SEC substrates beyond RGA, highlighting the broad implication of protein glycosylation in regulating cellular functions. In order to overcome the potential pleotropic effects brought by spy and sec mutations in planta and decipher the functional consequences of O-fucosylation and O-GlcNAcylation for specific target proteins, we will develop nanobody-fused SPY and SEC variants for precise target modification. Collectively, our study will fill the critical knowledge gap about the structural and functional consequences of protein glycosylation in plants. As SPY-like genes are conserved in diverse organisms, including plants, bacteria, and parasitic protists, and as SEC-like (OGT) genes are broadly distributed in plants and animals, a molecular understanding of these enzymes and their distinct structural and functional consequences will have a profound impact in plant biology, agriculture, and human health and diseases.