Elucidating the signaling and protein interaction networks of the O-GlcNAc transferase during embryonic stem cell state transitions

项目概要/摘要 O-GlcNAc是与细胞核和细胞质的丝氨酸和苏氨酸残基偶联的单个N-乙酰葡糖胺， proteins.与磷酸化类似，O-GlcNAc信号传导是动态的，快速添加和去除， 蛋白质以位点特异性方式响应细胞扰动和细胞外信号。因为两 修饰发生在相同的残基上，假设O-之间存在功能性串扰。 GlcNAc和磷酸化，其中一个可能影响另一个的沉积或去除。与磷酸化不同， 然而，这是由超过500激酶和大约300磷酸酶催化，哺乳动物的基因组只有 编码单个O-GlcNAc转移酶（OGT）和单个水解酶（OGA）。虽然许多激酶识别 由于其底物中存在特定的氨基酸序列，因此指导OGT的决定因素尚不清楚，可能是多种多样的。 这种细胞内糖基化几乎涉及从基因表达和信号传导到细胞内的每一个过程。 转导至细胞分裂和分化。尽管这种翻译后的 OGT在健康和疾病中的作用，OGT的特殊功能和O-GlcNAc信号传导的基本原理 仍然几乎完全难以捉摸。我们知识上的这种差距主要是由于缺乏工具， 研究O-GlcNAc信号传导或干扰基本OGT的技术。 在这里，我们的目标是揭示OGT和O-GlcNAc信号转导的基本原理，以及它们在转录调控中的作用。 调节细胞分化。我们最近开发了一种高灵敏度和特异性的浓缩 用于通过质谱法分析来自细胞和组织的O-GlcNAc修饰的肽的试剂。使用这些新 抗O-GlcNAc抗体，我们将阐明O-GlcNAc信号传导的全局，位点特异性时间动力学 在从全能性到幼稚和引发的多能性的转变期间。结合磷酸蛋白质谱 我们将监测这两种翻译后修饰之间的串扰。获得 深入了解OGT如何针对其不同的基板阵列，我们将去卷积广泛的OGT相互作用组 采用化学交联和生物化学分级分离，然后进行质谱分析。到 探索OGT如何使用衔接蛋白靶向底物，我们将使用一种创新的方法， 特异性OGT相互作用蛋白，并使用我们的新方法评估下游O-GlcNAc信号传导的变化 定量糖蛋白质组学方法。结合这两个研究项目，我们将创建一个整体的，高- 解析O-GlcNAc信号的原理。MIRA机制不仅可以使 研究基础O-GlcNAc生物学，但将提供灵活性，进行数据驱动的后续行动，功能 动态O-GlcNAc/串扰位点和不同OGT复合物的分析，以及它们在转录调控中的作用 一些最早的发展决策的监管。

Project Summary/Abstract O-GlcNAc is a single N-acetylglucosamine coupled to serine and threonine residues of nuclear and cytoplasmic proteins. Analogous to phosphorylation, O-GlcNAc signaling is dynamic, rapidly added and removed from proteins in a site-specific manner in response to cellular perturbations and extracellular cues. Because both modifications occur on the same residues it is hypothesized that there is a functional crosstalk between O- GlcNAc and phosphorylation, where one may affect deposition or removal the other. Unlike phosphorylation, however, which is catalyzed by over 500 kinases and roughly 300 phosphatases, the mammalian genome only encodes a single O-GlcNAc transferase (OGT) and a single hydrolase (OGA). While many kinases recognize specific amino acid sequences in their substrates, the determinants guiding OGT are unclear and likely manifold. This intracellular glycosylation is implicated in nearly every cellular process from gene expression and signal transduction to cell division and differentiation. Despite the ubiquitous nature of this post-translational modification in health and disease, the specific functions of OGT and the basic principles of O-GlcNAc signaling remain almost entirely elusive. This gap in our knowledge has been largely due to the major lack of tools and technologies available to study O-GlcNAc signaling or perturb the essential OGT. Here, we aim to uncover the basic principles of OGT and O-GlcNAc signaling, and their role in transcriptional regulation of cellular differentiation. We have recently developed a highly sensitive and specific enrichment reagent to analyze O-GlcNAc-modified peptides from cells and tissues by mass spectrometry. Using these new anti-O-GlcNAc antibodies we will elucidate the global, site-specific temporal dynamics of O-GlcNAc signaling during the transition from totipotency to naïve and primed pluripotency. Combined with phosphoprotemic profiling of the same samples, we will monitor for crosstalk between these two post-translational modifications. To gain insight into how OGT targets its diverse array of substrates, we will deconvolute the extensive OGT interactome employing chemical crosslinking and biochemical fractionation, followed by mass spectrometric analysis. To explore how OGT uses adaptor proteins to targets substrates, we will use an innovative approach, degrading specific OGT interacting proteins and assessing changes in downstream O-GlcNAc signaling using our new quantitative glycoproteomic approach. Integrating these two research programs, we will create a holistic, high- resolution understanding of the principles of O-GlcNAc signaling. The MIRA mechanism will not only enable the investigation of basic O-GlcNAc biology, but will provide the flexibility to conduct data-driven follow-up, functional analyses of dynamic O-GlcNAc/crosstalk sites and distinct OGT complexes, and their role in transcriptional regulation of some of the earliest development decisions.