Photocrosslinking probes to discover glycan-dependent interactions

光交联探针发现聚糖依赖性相互作用

• 批准号: 9166533
• 负责人: Jennifer J Kohler
• 金额: $ 31.41万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-25 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9166533
• 关键词: Affect; Affinity; Alzheimer' s Disease; Binding; Biochemistry; Biological; Biological Assay; Biological Models; Biological Process; Biology; Cardiovascular Diseases; Cell Line; Cells; Chemicals; Communities; Complex; Core Facility; Covalent Interaction; Cytoplasmic Protein; Development; Diabetes Mellitus; Diazomethane; Disease; Ensure; Environment; Glycopeptides; Goals; Housing; Immunoblotting; In Vitro; Interest Group; Laboratories; Letters; Life; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Medical center; Methods; Modification; Neurodegenerative Disorders; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Pore Complex Proteins; Nuclear Proteins; Outcome; Peptide Synthesis; Peptides; Plant Proteins; Plasmids; Polysaccharides; Post-Translational Protein Processing; Procedures; Proteins; Proteomics; Protocols documentation; Reagent; Reporting; Research; Role; Stimulus; Stress; System; Vascular Plant; Work; Workplace; base; biomedical scientist; candidate identification; cellular engineering; crosslink; functional group; improved; innovation; insight; method development; nanoparticle; novel; protein function; response; tool; ultraviolet irradiation

The goal of this project is to develop accessible and effective methods to discover the interaction partners of proteins that are modified with O-linked beta-N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a common post- translational modification of intracellular proteins in metazoa and higher plants. Hundreds of O-GlcNAc- modified nuclear and cytoplasmic proteins have been identified. Although O-GlcNAc is both abundant and essential, little is known about how it affects the function of modified proteins. The central hypothesis of the proposed work is that O-GlcNAc-modified proteins are surrounded by a different set of molecules than the unmodified proteins. Obtaining information about the interaction partners of O-GlcNAc-modified proteins will provide new insight into O-GlcNAc function. To identify O-GlcNAc interaction partners, we will rely on a photocrosslinking approach to convert low-affinity glycan-dependent interactions to high-affinity covalent complexes that can be purified and characterized by mass spectrometry. Two complementary approaches will be pursued: an in-lysate photocrosslinking approach (Aim 1) and an in-cell photocrosslinking approach (Aim 2). In the first aim, a panel of photocrosslinking glycopeptide probes will be prepared. These molecules will be crosslinked to proteins in lysate. The crosslinked complexes will be isolated and the interaction partners will be identified by mass spectrometry. The outcome of this aim will be an in-lysate photocrosslinking method that has the following features: (a) probe molecules produced by routine, simple methods, (b) probe molecules crosslinked efficiently and specifically to interacting proteins, (c) LC-MS/MS delivering a high yield of tryptic peptides from a specific subset of proteins, and (d) candidate interacting proteins confirmed by in vitro and cell-based assays. In the second aim, a photocrosslinking functional group will be incorporated onto O-GlcNAc residues in living cells. Subsequent UV irradiation will result in covalent crosslinking of O-GlcNAc-associated interactions in their native cellular context. As in Aim 1, the crosslinked complexes will be isolated and the interaction partners will be identified by mass spectrometry. The basic features of this in-cell photocrosslinking method have already been reported; the aim of this proposal is to make the method more effective and powerful by (a) engineering cells to produce more O-GlcNDAz, (b) purifying crosslinked material more efficiently and stringently, and (c) improving confidence in candidate hit identification. The reagents and methods developed will be shared with other research groups to enable study of a wide variety of O-GlcNAc- modified proteins with diverse biological functions. The proposed work places a high priority on 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 O-GlcNAc is associated with multiple disease states including type II diabetes, cardiovascular disease, Alzheimer's disease, and several cancers.

该项目的目的是开发可访问有效的方法，以发现 用O连锁β-N-乙酰葡萄糖胺（O-GLCNAC）修饰的蛋白质。 O-GLCNAC是一个常见的后 转化的细胞内蛋白质和高等植物中的细胞内蛋白质。数百个O-glcnac- 已经鉴定出修饰的核和细胞质蛋白。虽然O-GlcNAC既丰富又 对于它如何影响修饰蛋白的功能，重要的是，知之甚少。中心假设 拟议的工作是O-GlCNAC修饰的蛋白质与不同的分子围绕着 未改性的蛋白质。获取有关O-GLCNAC修饰蛋白的相互作用伙伴的信息 将提供有关O-GLCNAC功能的新见解。要识别O-GLCNAC互动伙伴，我们将依靠 将低亲和力聚糖依赖性相互作用转换为高亲和力共价的光链接方法 可以通过质谱法纯化和表征的复合物。两种完整的方法将 被追捕：一种赖以生殖的光链接方法（AIM 1）和一个内部的光链接方法（AIM 2）。在第一个目标中，将准备一系列光叠型糖肽探针。这些分子将是 在裂解物中交联至蛋白质。交联的复合物将被隔离，相互作用伙伴将 通过质谱法识别。这个目标的结果将是一种赖以生态的光链接方法 具有以下特征：（a）通过常规，简单方法产生的探针分子，（b）探针分子 有效，专门针对相互作用的蛋白质交联，（c）LC-MS/MS提供高胰蛋白酶的产量 来自特定蛋白质的特定子集的肽，以及（d）通过体外和 基于细胞的测定。在第二个目的中，将在O-GLCNAC上纳入光链接功能组 活细胞中的残留物。随后的紫外线照射将导致O-GLCNAC相关的共价交联 在其天然细胞环境中的相互作用。与AIM 1一样，交联的复合物将被隔离，并 相互作用伙伴将通过质谱识别。此室内的基本功能 已经报道了Photocropslinking方法；该提议的目的是使方法更加 （a）工程单元生成更多的O-Glcndaz，（b）净化交联材料的有效和强大 更有效，更严格，以及（c）提高对候选命中识别的信心。试剂和 开发的方法将与其他研究小组共享，以研究各种O-GlcNAC- 具有潜水生物学功能的修饰蛋白质。拟议的工作将高度重视 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界非常重要，因为O-GLCNAC的失调与 具有多种疾病状态，包括II型糖尿病，心血管疾病，阿尔茨海默氏病和几种 癌症。