Role of O-GlcNAc in Metabolic Signaling

描述（由申请人提供）：O-linked β - 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，共价附着在细胞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，转移到细胞内蛋白的O-GlcNAc水平升高可诱导胰岛素抵抗，这是II型糖尿病的标志。本提案的具体目的是验证胰岛素作用被胰岛素信号转导级联中特定蛋白的O-GlcNAc修饰升高所抑制的假设。在3种不同的模型系统中，我们已经确定缺陷位于胰岛素信号的代谢分支，受体的下游以及蛋白激酶AKT的上游或上游。我们已经开发了几个必要的工具来研究O-GlcNAc在胰岛素信号转导中的翻译后修饰。其中包括O-GlcNAc特异性单克隆抗体，添加和去除O-GlcNAc的酶的诱导表达细胞系，O-GlcNAc循环酶缺失的秀丽隐杆线虫菌株，以及基于串联质谱的位点定位和翻译后修饰定量方法。在Specific Aim 1中，我们将扩展我们的初步研究结果，以哺乳动物细胞系和秀丽隐杆线虫为模型系统，通过细胞凋亡、葡萄糖摄取和/或寿命的变化来证明，不适当的O-GlcNAc修饰会干扰胰岛素信号传导。此外，在全球O-GlcNAc水平受到扰动时，将阐明胰岛素途径代谢分支蛋白的功能变化（活性、定位、关联和磷酸化）。在Specific Aim 2中，我们将使用我们的串联质谱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Specific Aim 3中，我们将研究O-GlcNAc修饰胰岛素级联代谢分支中特定信号转导蛋白的功能后果。通过将标记的具有糖基化能力的野生型和无糖基化能力的突变蛋白重新引入哺乳动物细胞系和线虫菌株，我们将确定特定蛋白上特定位点的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秀丽隐杆线虫中调节胰岛素作用方面的全局和分子后果。

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.