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

项目摘要/摘要 O-GlcNAc是一种单一的N-乙酰氨基葡萄糖，偶联于细胞核和细胞质中的丝氨酸和苏氨酸残基 蛋白质。与磷酸化类似，O-GlcNAc信号是动态的，可以快速添加和删除 蛋白质以特定部位的方式对细胞扰动和细胞外信号做出反应。因为两者都 修饰发生在相同的残基上，假设O-之间存在功能串扰 GlcNAc和磷酸化，其中一个可能影响沉积或去除另一个。与磷酸化不同， 然而，这是由500多个激酶和大约300个磷酸酶催化的，只有哺乳动物的基因组 编码单一的O-GlcNAc转移酶(OGT)和单一的水解酶(OGA)。当许多激酶识别出 在其底物中的特定氨基酸序列中，指导OGT的决定因素尚不清楚，可能有多种。 这种细胞内的糖基化参与了几乎所有的细胞过程，从基因表达到信号。 转导至细胞分裂和分化。尽管这种后翻译的性质无处不在 健康与疾病的修饰、OGT的特殊功能和O-GlcNAc信号的基本原理 几乎完全难以捉摸。我们知识上的这种差距在很大程度上是由于缺乏工具和 可用于研究O-GlcNAc信号或扰乱基本OGT的技术。 在这里，我们旨在揭示OGT和O-GlcNAc信号的基本原理，以及它们在转录中的作用 细胞分化的调控。我们最近开发了一种高度敏感和特异的富集物 细胞和组织中O-GlcNAc修饰多肽的质谱分析试剂。使用这些新功能 抗O-GlcNAc抗体我们将阐明O-GlcNAc信号的全球、特定部位的时间动力学 在从全能到天真和准备好的全能的过渡过程中。结合磷蛋白图谱分析 对于相同的样本，我们将监测这两个翻译后修改之间的串扰。为了获得 深入了解OGT如何瞄准其多样化的底物阵列，我们将揭开OGT广泛的互动基因组 采用化学交联法和生化分级法，然后进行质谱分析。至 探索OGT如何使用接头蛋白来靶向底物，我们将使用一种创新的方法，降解 特定的OGT相互作用蛋白和使用我们的新技术评估下游O-GlcNAc信号的变化 定量糖蛋白组学方法。将这两个研究项目结合起来，我们将创造一个全面、高度- 了解O-GlcNAc信号的原理。米拉机制不仅将使 调查基本的O-GlcNAc生物学，但将提供进行数据驱动的后续功能的灵活性 动态O-GlcNAc/串扰位点和不同OGT复合体的分析及其在转录中的作用 对一些最早的开发决策进行监管。

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.