New tools for studying GlcNAc biology

研究 GlcNAc 生物学的新工具

基本信息

批准号：
9814544
负责人：
Jennifer J Kohler
金额：
$ 50.38万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9814544
关键词：
Adoption Alkynes Area Binding Binding Sites Biological Biological Assay Biological Process Biology Cancer Biology Carbohydrates Cells Chemicals Chemistry Collaborations Communities Complex Computer software Crosslinker Cytolysis Data Developmental Biology Diabetes Mellitus Diazomethane Disease Enzymes Generations Glycobiology Glycoconjugates Goals Health Hexosamines Human Immunology Infectious Diseases Research Introns Label Link Lipids Malignant Neoplasms Mass Spectrum Analysis Measures Metabolic Metabolism Methodology Methods Modification Molecular Monitor Monosaccharides N-Acetylglucosaminyltransferases Nerve Degeneration Neurodegenerative Disorders Nucleotides O-GlcNAc transferase Pathology Pathway interactions Plasmids Play Polysaccharides Positioning Attribute Procedures Production Proteins Protocols documentation Publications RNA Splicing Reagent Recombinants Reporter Reporting Research Research Personnel Role Serine Signal Pathway Signal Transduction Signal Transduction Pathway Site Structure Synthesis Chemistry Techniques Technology Threonine Transcript Transferase Work analog base crosslink detection of nutrient experience extracellular glycoproteomics glycosylation human disease improved insight mutant protein crosslink sugar tool

项目摘要

Project Summary/Abstract The goal of this project is to develop accessible and effective methods to monitor the levels and interaction partners of GlcNAc-containing glycoconjugates. N-acetylglucosamine (GlcNAc) is a monosaccharide found in many classes of mammalian glycoconjugates. Addition of GlcNAc to serine and threonine residues forms the intracellular O-GlcNAc modification, and GlcNAc is also incorporated into many extracellular glycoconjugates including N-linked glycans and GalNAc-type glycans. Incorporation of GlcNAc into glycoconjugates is catalyzed by the activity of N-acetylglucosaminyltransferases (GlcNAc-transferases) that transfer GlcNAc from the donor UDP-GlcNAc to (glyco)protein and (glyco)lipid acceptors. UDP-GlcNAc is produced through the nutrient-sensing hexosamine biosynthetic pathway, which integrates information about carbohydrate, protein, lipid, and nucleotide availability. UDP-GlcNAc levels regulate production of key glycan structures, namely O- GlcNAcylation and N-linked glycan branching, which in turn control essential signal transduction pathways. Thus, GlcNAc-containing glycans represent a crucial link between metabolic state and cellular signaling. However, cell-based methods to characterize the levels and interaction partners of these molecules remain inadequate. Aim 1 will deliver non-invasive, non-perturbing fluorescent and luminescent reporters of intracellular O-GlcNAc levels. This Aim builds on the discovery that splicing of the O-GlcNAc transferase (OGT) transcript responds rapidly to changes in O-GlcNAc levels. Aims 2-4 improve upon previously reported photocrosslinking sugar technology, in which the diazirine photoactivatable crosslinking group is installed on GlcNAc residues in living cells. Aim 2 will make this technology easier to use by simplifying the reagents, improving crosslinking yield, and facilitating purification of crosslinked complexes. Aim 3 will make this technology broader in scope by introducing photocrosslinking GlcNAc into additional classes of glycoconjugates, including N-linked glycans. Aim 4 will make the technology more powerful by developing a mass spectrometry strategy to identify not only the identity of the sites of the binding partners, but also the sites of crosslinking. The mass spectrometry-based approach to crosslinking analysis will capture molecular details of O-GlcNAc-dependent interactions that occur in living cells. The reagents and methods developed in this proposal will be shared with other research groups to enable study of a wide variety of O-GlcNAcylation and N-glycosylated proteins with diverse biological functions. The proposed work prioritizes approaches that are simple to implement and make use of “off-the-shelf” reagents and procedures. Making these methods available to the broad biomedical community is significant because dysregulation of GlcNAc-containing glycoconjugates is associated with multiple disease states including diabetes, neurodegenerative disease, and cancer.

项目概要/摘要该项目的目标是开发可访问且有效的方法来监测水平和相互作用含有 GlcNAc 的糖复合物的伙伴是 N-乙酰葡糖胺 (GlcNAc) 是一种单糖。许多类别的哺乳动物糖缀合物。GlcNAc 添加至丝氨酸和苏氨酸残基形成了细胞内 O-GlcNAc 修饰，并且 GlcNAc 也掺入许多细胞外糖缀合物中包括 N 连接聚糖和 GalNAc 型聚糖将 GlcNAc 掺入糖复合物中。由 N-乙酰氨基葡萄糖转移酶（GlcNAc 转移酶）的活性催化，将 GlcNAc 从 (糖)蛋白和(糖)脂质受体的供体UDP-GlcNAc是通过以下过程产生的。营养感应己糖胺生物合成途径，整合了碳水化合物、蛋白质、脂质和核苷酸的可用性调节关键聚糖结构（即 O-）的产生。 GlcNA 酰化和 N 连接聚糖分支，进而控制重要的信号转导途径。因此，含有 GlcNAc 的聚糖代表了代谢状态和细胞信号传导之间的关键联系。然而，基于细胞的方法来表征这些分子的水平和相互作用伙伴仍然存在目标 1 将提供非侵入性、非扰动的荧光和发光产生器。该目标建立在 O-GlcNAc 转移酶剪接的发现之上。 (OGT) 转录物对 O-GlcNAc 水平的变化做出快速反应，目标 2-4 比之前报道的有所改进。光交联糖技术，其中二氮丙啶光活化交联基团安装在活细胞中的 GlcNAc 残留物将通过简化试剂使该技术更易于使用，提高交联产率并促进交联复合物的纯化将实现这一目标。通过将光交联 GlcNAc 引入其他类别，技术范围更加广泛糖缀合物，包括 N-连接聚糖，Aim 4 将通过开发一种更强大的技术。质谱策略不仅可以识别结合伙伴位点的身份，还可以识别基于质谱的交联分析方法将捕获分子。活细胞中发生的 O-GlcNAc 依赖性相互作用的详细信息。该提案将与其他研究小组共享，以便能够研究各种 O-GlcNAcylation 以及具有多种生物功能的 N-糖基化蛋白质。实施这些方法很简单，并且可以使用“现成的”试剂和程序。由于 GlcNAc 的失调，对广泛的生物医学界来说是重要的糖复合物与多种疾病状态相关，包括糖尿病、神经退行性疾病和癌症。