Role of O-GlcNAc in Metabolic Signaling

描述（由申请人提供）：O-连接β-N-乙酰葡糖胺（O-GlcNAc）是一种动态和诱导型翻译后修饰，与细胞核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，转移到细胞内蛋白质的O-GlcNAc水平的升高诱导胰岛素抵抗，这是II型糖尿病的标志。本提案的具体目的是检验胰岛素作用受胰岛素信号转导级联中特定蛋白质的O-GlcNAc修饰升高抑制的假设。在3个不同的模型系统中，我们已经确定，缺陷在于胰岛素信号的代谢分支，受体的下游和蛋白激酶AKT的上游或下游。我们已经开发了几个必不可少的工具，研究O-GlcNAc在胰岛素信号转导中的翻译后修饰。这些包括O-GlcNAc特异性单克隆抗体、用于诱导表达添加和去除O-GlcNAc的酶的细胞系、C. elegans菌株中的O-GlcNAc循环酶，和串联质谱为基础的方法，位点定位和定量的翻译后修饰。在具体目标1中，我们将扩展我们的初步研究结果，以证明不适当的O-GlcNAc修饰干扰胰岛素信号传导，通过使用哺乳动物细胞系和C. elegans作为模型系统。此外，还将阐明总体O-GlcNAc水平扰动后胰岛素途径代谢分支蛋白质的功能变化（活性、定位、关联和磷酸化）。在具体目标2中，我们将使用我们的串联质谱法为基础的方法来识别和定位与胰岛素信号通路相关的O-GlcNAc修饰蛋白。在具体目标3中，我们将对胰岛素级联的代谢分支中特定信号转导蛋白的O-GlcNAc修饰分配功能性后果。通过将标记的糖基化活性野生型和糖基化活性缺失突变蛋白重新导入哺乳动物细胞系和C。通过对感兴趣的蛋白质为零的线虫菌株进行筛选，我们将确定特定蛋白质上的特定位点的差异O-GlcNAc修饰如何影响胰岛素信号传导。这些目标的完成将阐明蛋白O-GlcNAc修饰在哺乳动物细胞系和C.优雅

DESCRIPTION (provided by applicant): O-linked beta-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 3 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 posttranslational 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 sitemap 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.