Role of O-GIcNAc in Metabolic Signaling

O-连接的（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 其共价连接到细胞核和细胞质蛋白上的丝氨酸和苏氨酸残基。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 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检验 胰岛素作用受特定蛋白质O-GlcNAc修饰升高抑制的假说 在胰岛素信号转导级联中。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传导的代谢分支中，在受体的下游和在 蛋白激酶AKT。我们已经开发了几个必要的工具，用于研究O-GlcNAc后， 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， 抗体、用于诱导表达添加和去除O-GlcNAc的酶的细胞系、C. elegans 在O-GlcNAc循环酶中具有缺失的菌株，以及基于串联质谱的 翻译后修饰的位点定位和定量方法。 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，如通过细胞凋亡、葡萄糖摄取和/或寿命的变化所测量的 使用哺乳动物细胞系和C. elegans作为模型系统。此外，功能变化（活动， 定位、结合和磷酸化）在胰岛素途径的代谢分支的蛋白质中 将阐明对总体O-GlcNAc水平扰动的影响。在具体目标2中，我们将确定和定位- 使用我们的串联图谱， 基于质谱的方法。在具体目标3中，我们将把功能性后果分配给O- 胰岛素级联反应代谢分支中特异性信号转导蛋白的GlcNAc修饰。 通过重新引入带标签的糖基化活性野生型和糖基化活性缺失突变蛋白 回到哺乳动物细胞系和C.对于感兴趣的蛋白质无效的线虫菌株，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 信号这些目标的完成将阐明蛋白质O的全局和分子后果。 在哺乳动物细胞系和C.优雅

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.