New tools for studying GlcNAc biology

研究 GlcNAc 生物学的新工具

• 批准号: 10187532
• 负责人: Jennifer J Kohler
• 金额: $ 48.83万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10187532
• 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

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.

项目摘要/摘要 该项目的目的是开发可访问有效的方法来监视水平和交互 含GlcNAC的糖缀合物的合作伙伴。 N-乙酰葡萄糖（GLCNAC）是一种单糖，发现 许多类别的哺乳动物糖缀合物。在丝氨酸和苏氨酸残留物中添加GlcNAC形成 细胞内O-GLCNAC修饰，GlcNAC也被纳入许多细胞外糖缀合物 包括N连接的聚糖和Galnac型聚糖。将GlcNAC掺入糖缀合物为 由N-乙酰葡萄糖氨基转移酶（GlcNAC-转移酶）的活性催化 供体UDP-GLCNAC为（Glyco）蛋白和（Glyco）脂质受体。 UDP-GLCNAC是通过 营养敏感性六胺生物合成途径，该途径整合了有关碳氢，蛋白质， 脂质和核苷酸的可用性。 UDP-GLCNAC水平调节关键聚糖结构的产生，即O- Glcnacylation和N连接的聚糖分支，进而控制基本信号转导途径。 这，含GlcNAC的聚糖代表了代谢状态与细胞信号传导之间的关键联系。 但是，基于细胞的方法来表征这些分子的水平和相互作用伙伴 AIM 1将提供无创，非扰动荧光和发光记者 细胞内O-GLCNAC水平。这个目标是基于发现O-GLCNAC转移酶的发现 （OGT）转录本对O-GLCNAC水平的变化的响应迅速。 AIMS 2-4改进了先前报道的 Photocropslinking糖技术，在该技术中安装了重氮嗪光活化的交联组 GlcNAC保留在活细胞中。 AIM 2将通过简化试剂，使该技术更易于使用， 提高交联产量，并支持交联配合物的纯化。 AIM 3将成为这个 通过将GlcNAC引入其他类别 糖缀合物，包括N连接的聚糖。 AIM 4将通过开发一个 质谱策略不仅确定结合伙伴位置的身份，还确定 交联站点。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依赖性相互作用的细节。试剂和方法在 该建议将与其他研究小组共享，以实现各种O-Glcnacylation的研究 具有潜水生物学功能的N-糖基化蛋白质。拟议的工作优先考虑 易于实施并利用“现成”试剂和程序。制作这些方法 可用于广泛的生物医学群落是重要的，因为含GlcNAC的失调 糖缀合物与多种疾病状态有关，包括糖尿病，神经退行性疾病和 癌症。

项目成果

Photocrosslinking O-GlcNAcylated Proteins to Neighboring Biomolecules.

• DOI: 10.1002/cpz1.201
• 发表时间: 2021-07
• 期刊: Current protocols
• 作者: Capota E;Wu H;Kohler JJ
• 通讯作者: Kohler JJ

What sugar does to your pores.

• DOI: 10.1083/jcb.202105163
• 发表时间: 2021-07-05
• 期刊: The Journal of cell biology
• 作者: Kohler JJ