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

阐明胚胎干细胞状态转变过程中 O-GlcNAc 转移酶的信号传导和蛋白质相互作用网络

• 批准号: 10659048
• 负责人: Sam Anthony Myers
• 金额: $ 45.75万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659048
• 关键词: Adaptor Signaling Protein; Affect; Amino Acid Sequence; Antibodies; Biochemical; Biological Process; Biology; Cell Differentiation process; Cell division; Cell physiology; Cells; Chemicals; Complex; Coupled; Cues; Cytoplasmic Protein; Data; Deposition; Development; Disease; Excision; Fractionation; Gene Expression; Gene Expression Process; Health; Hydrolase; Investigation; Knowledge; Mammals; Mass Spectrum Analysis; Modification; Monitor; Nature; Nuclear Proteins; O-GlcNAc transferase; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Proteins; Reagent; Research; Resolution; Role; Sampling; Serine; Signal Pathway; Signal Transduction; Site; Technology; Threonine; Tissues; Totipotency; Transcriptional Regulation; cell growth regulation; crosslink; embryonic stem cell; extracellular; flexibility; follow-up; glycoproteomics; glycosylation; innovation; insight; mammalian genome; pluripotency; programs; response; stem cell differentiation; tool

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.

项目概要/摘要 O-GlcNAc 是与核和细胞质的丝氨酸和苏氨酸残基偶联的单个 N-乙酰氨基葡萄糖 与磷酸化类似，O-GlcNAc 信号传导是动态的，可以快速添加和去除。 蛋白质以位点特异性方式响应细胞扰动和细胞外信号。 修饰发生在相同的残基上，概括地说，O-之间存在功能串扰 GlcNAc 和磷酸化，其中一个可能影响另一个的沉积或去除， 然而，哺乳动物基因组仅由 500 多种激酶和大约 300 种磷酸酶催化 编码单个 O-GlcNAc 转移酶 (OGT) 和单个水解酶 (OGA)，而许多激酶可识别。 由于其底物中的特定氨基酸序列，指导 OGT 的决定因素尚不清楚，而且可能是多种多样的。 这种细胞内糖基化涉及基因表达和信号的几乎所有细胞过程 尽管这种翻译后的性质普遍存在。 健康和疾病的改变、OGT 的具体功能以及 O-GlcNAc 信号传导的基本原理 我们的知识差距几乎完全难以捉摸，这主要是由于工具和工具的严重缺乏。 可用于研究 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 信号传导的基本原理，以及它们在转录中的作用 我们最近开发了一种高度灵敏和特异性的富集方法。 使用这些新试剂来分析细胞和组织中的 O-GlcNAc 修饰肽。 抗 O-GlcNAc 抗体，我们将阐明 O-GlcNAc 信号传导的全局、位点特异性时间动态 从全能性到幼稚和引发多能性的转变过程中与磷酸化蛋白质分析相结合。 对于相同的样品，我们将监测这两个翻译后修饰之间的串扰以获得。 深入了解 OGT 如何靶向其多种底物，我们将对广泛的 OGT 相互作用组进行解卷积 采用化学交联和生化分级分离，然后进行质谱分析。 探索OGT如何利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 相互作用蛋白并使用我们的新方法评估下游 O-GlcNAc 信号传导的变化 整合这两个研究项目，我们将创建一个整体的、高水平的糖蛋白组学方法。 了解 O-GlcNAc 信号传导原理的分辨率不仅能够实现 MIRA 机制。 对基本 O-GlcNAc 生物学的研究，但将提供灵活性来进行数据驱动的后续、功能 分析动态 O-GlcNAc/串扰位点和不同的 OGT 复合物及其在转录中的作用 监管一些最早的发展决策。

项目成果

Proteome-wide base editor screens to assess phosphorylation site functionality in high-throughput.

全蛋白质组碱基编辑器筛选以评估高通量磷酸化位点功能。

• 发表时间: 2023-11-14
• 作者: Kennedy, Patrick H;Deh Sheikh, Amin Alborzian;Balakar, Matthew;Jones, Alexander C;Olive, Meagan E;Hegde, Mudra;Matias, Maria I;Pirete, Natan;Burt, Rajan;Levy, Jonathan;Little, Tamia;Hogan, Patrick G;Liu, David R;Doench, John G;Newton, Alexan
• 通讯作者: Newton, Alexan