Role of O-GIcNAc in Metabolic Signaling

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 其他研究表明，O-GlcNAc水平的升高转移到细胞内蛋白质 导致胰岛素抵抗，这是II型糖尿病的标志。这项提案的具体目标将受到考验 胰岛素作用受特定蛋白质O-GlcNAc修饰抑制的假说 在胰岛素信号转导的级联反应中。在三个不同的模型系统中，我们已经确定 缺陷存在于胰岛素信号的代谢分支，位于受体下游，位于或位于 蛋白激酶AKT。我们已经开发了几个必要的工具来研究O-GlcNAc后- 胰岛素信号转导中的翻译修饰。其中包括O-GlcNAc特异性单抗 抗体，可诱导表达添加和去除O-GlcNAc、线虫的酶的细胞系 含有O-GlcNAc循环酶缺失的菌株，以及基于串联质谱仪的 翻译后修改的现场测绘和量化的方法。 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 通过细胞凋亡、葡萄糖摄取和/或寿命的变化来衡量修饰对胰岛素信号转导的影响 以哺乳动物细胞系和线虫为模型系统。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 关于全球O-GlcNAc水平的扰动，将被阐明。在具体目标2中，我们将确定并确定- MAP O-GlcNAc修饰的与胰岛素信号通路相关的蛋白质 基于质谱学的方法。在具体目标3中，我们将为O-分配函数结果 胰岛素级联代谢分支中特定信号转导蛋白的GlcNAc修饰。 通过重新引入标记糖基化能力的野生型和糖基化不能力的突变蛋白 回到哺乳动物细胞系和对感兴趣的蛋白质无效的线虫品系中，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-蛋白的全球和分子后果。 在哺乳动物细胞系和线虫中调节胰岛素作用的GlcNAc修饰。

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.