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：
7336329
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/7336329
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 Elevation 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 Overexpression Protein-Serine-Threonine Kinases Proteins Proto-Oncogene Proteins c-akt Rate 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 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连接的（3-N-乙酰葡萄糖胺（O- GlcNAC）是一种动态且可诱导的翻译后修饰共价连接到核和细胞质蛋白上的丝氨酸和苏氨酸残基上。我们和其他人已经证明，O-GLCNAC水平的升高转移到细胞内蛋白诱导胰岛素抵抗，这是II型糖尿病的标志。该提案的具体目的将测试特异性蛋白的O-GLCNAC修饰抑制了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，我们确定了缺陷位于胰岛素信号传导的代谢分支，受体的下游以及在或上游蛋白激酶Akt。我们为研究O-GLCNAC后的研究开发了几种基本工具。胰岛素信号转导中的翻译修饰。这些包括O-GLCNAC特异性单克隆抗体，用于添加和去除O-GlCNAC的酶诱导表达的细胞系，秀丽隐杆线虫在O-GLCNAC循环酶中删除的菌株和基于串联质谱的菌株用于翻译后修改的站点映射和量化的方法。在特定目标1中，我们将扩展我们的初步发现，以证明不适当的O-GLCNAC 修饰通过细胞凋亡，葡萄糖摄取和/或寿命的变化来衡量的胰岛素信号传导使用哺乳动物细胞系和秀丽隐杆线虫作为模型系统。此外，功能变化（活动，在胰岛素途径的代谢分支的蛋白质中定位，关联和磷酸化）全球O-GLCNAC水平的扰动将被阐明。在特定的目标2中，我们将确定和站点 - MAP O-GLCNAC修饰的蛋白与胰岛素信号通路相关的蛋白使用我们的串联套件基于质谱的方法。在特定的目标3中，我们将为O-分配功能后果胰岛素级联代谢分支中特定信号转导蛋白的GLCNAC修饰。通过重新引入标记的糖基化功能型野生型和糖基化不足的突变蛋白回到哺乳动物细胞系和秀丽隐杆线虫菌株中无效的蛋白质，我们将确定特定蛋白质上特定位点的差异O-GLCNAC如何影响胰岛素信号。这些目标的完成将阐明蛋白O-的全球和分子后果 GlcNAC修饰在调节哺乳动物细胞系和秀丽隐杆线虫中的胰岛素作用方面。