Role of O-GIcNAc in Metabolic Signaling

O-GlcNAc 在代谢信号传导中的作用

基本信息

批准号：
7545835
负责人：
Lance Wells
金额：
$ 26.74万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-01-01 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7545835
关键词：
1-Phosphatidylinositol 3-Kinase Acetylglucosamine Adipocytes Adult Affect Americas Amputation Antibodies Apoptosis Attenuated Back Biological Models Blindness Caenorhabditis elegans Cell Line Chinese Hamster Ovary Cell Cytoplasmic Protein Defect Deletion Mutation Development Disease Enzymes Epitopes Event Excision Genes Genetic Global Change Incidence Insulin Insulin Receptor Insulin Resistance Insulin Signaling Pathway Kidney Failure Link Longevity Mammalian Cell Maps Mass Spectrum Analysis Measures Mediating Metabolic Methodology Modification Molecular Monitor Monoclonal Antibodies Myocardial Infarction N acetylglucosaminidase Nematoda Non-Insulin-Dependent Diabetes Mellitus Nuclear O-GlcNAc transferase PDPK1 gene Pathway interactions Phenotype Phosphorylation Population Post-Translational Protein Processing Property Protein Glycosylation Protein Kinase Protein-Serine-Threonine Kinases Proteins Proto-Oncogene Proteins c-akt Rattus Receptor Cell Research Personnel Rodent Role Serine Signal Pathway Signal Transduction Signal Transduction Pathway Signaling Molecule Site Stroke Testing Threonine Transgenic Organisms United States base glucose uptake glycosylation insulin signaling interest member mutant overexpression peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase prevent programs receptor retinal neuron tandem mass spectrometry tool

项目摘要

O-linked (3-N-acetylglucosamine(O- GlcNAc) is a dynamic and inducible post-translational modification that is covalently attached to serine and threonine residues on nuclear and cytoplasmic proteins. We and others have demonstrated that elevation in the levels of O-GlcNAc transferred to intracellular proteins induces insulin resistance, the hallmark of Type II diabetes. The specific aims of this proposal will test the hypothesis that insulin action is inhibited by elevated O-GlcNAc modification of specific proteins in the insulin signal transduction cascade. In three different model systems, we have determined that the defect lies in the metabolic branch of insulin signaling, downstream of the receptor and at or upstream of the protein kinase AKT. We have developed several essential tools for the study of the O-GlcNAc post- translational modification in insulin signal transduction. These include an O-GlcNAc specific monoclonal antibody, cell lines for the inducible expression of enzymes that add and remove O-GlcNAc, C. elegans strains that harbor deletions in the O-GlcNAc cycling enzymes, and a tandem mass spectrometry-based approach for site-mapping and quantification of post-translational modifications. In Specific Aim 1, we will expand on our preliminary findings to demonstrate that inappropriate O-GlcNAc modification perturbs insulin signaling as measured by changes in apoptosis, glucose-uptake, and/or lifespan using mammalian cell lines and C. elegans as model systems. Furthermore, functional changes (activity, localization, associations, and phosphorylation) in proteins of the metabolic branch of the insulin pathway upon perturbations in global O-GlcNAc levels will be elucidated. In Specific Aim 2, we will identify and site- map O-GlcNAc modified proteins associated with the insulin signaling pathway using our suite of tandem mass spectrometry-based approaches. In Specific Aim 3, we will assign functional consequences to O- GlcNAc modification of specific signal transduction proteins in the metabolic branch of the insulin cascade. By reintroducing tagged glycosylation-competent wild type and glycosylation-incompetent mutant proteins back into mammalian cell lines and into C. elegans strains that are null for the protein of interest, we will determine how differential O-GlcNAc modification of specific sites on particular proteins affects insulin signaling. Completion of these aims will elucidate both global and molecular consequences of protein O- GlcNAc modification with regard to regulating insulin action in mammalian cell lines and in C. elegans.

O-连接（3-N-乙酰氨基葡萄糖（O-GlcNAc）是一种动态的、可诱导的翻译后修饰它与核蛋白和细胞质蛋白上的丝氨酸和苏氨酸残基共价连接。我们和其他人已经证明，O-GlcNAc 水平的升高会转移到细胞内蛋白质诱导胰岛素抵抗，这是 II 型糖尿病的标志。该提案的具体目标将测试特定蛋白质的 O-GlcNAc 修饰升高会抑制胰岛素作用的假设在胰岛素信号转导级联中。在三个不同的模型系统中，我们确定缺陷存在于胰岛素信号传导的代谢分支中，位于受体的下游以及受体的上游或下游。蛋白激酶 AKT。我们开发了几种用于 O-GlcNAc 后研究的重要工具。胰岛素信号转导的翻译修饰。其中包括 O-GlcNAc 特异性单克隆抗体抗体、用于添加和去除 O-GlcNAc 的酶诱导表达的细胞系、秀丽隐杆线虫含有 O-GlcNAc 循环酶缺失的菌株，以及基于串联质谱法的翻译后修饰的位点测绘和量化方法。在具体目标 1 中，我们将扩展我们的初步发现，以证明不适当的 O-GlcNAc 修饰会扰乱胰岛素信号传导（通过细胞凋亡、葡萄糖摄取和/或寿命的变化来衡量）使用哺乳动物细胞系和线虫作为模型系统。此外，功能变化（活动、胰岛素途径代谢分支蛋白质中的定位、关联和磷酸化）将阐明全球 O-GlcNAc 水平的扰动。在具体目标 2 中，我们将确定并定位- 使用我们的串联套件绘制与胰岛素信号通路相关的 O-GlcNAc 修饰蛋白基于质谱的方法。在具体目标 3 中，我们将功能后果分配给 O- GlcNAc 修饰胰岛素级联代谢分支中的特定信号转导蛋白。通过重新引入标记的具有糖基化能力的野生型和不具有糖基化能力的突变蛋白回到哺乳动物细胞系和线虫菌株中，这些菌株对感兴趣的蛋白质无效，我们将确定特定蛋白质上特定位点的差异 O-GlcNAc 修饰如何影响胰岛素发信号。这些目标的完成将阐明 O-蛋白的整体和分子后果 GlcNAc 修饰与调节哺乳动物细胞系和秀丽隐杆线虫中的胰岛素作用有关。