O-GlcNAc:Developing New Tool for Assessment of Glycemia

描述（由申请人提供）： 通过酶促连接O-连接的N-乙酰葡糖胺至丝氨酸或苏氨酸残基（O-GlcNAc）对核质蛋白的动态修饰现在已知是营养和应激传感器，调节细胞信号传导、转录、蛋白酶体活性和细胞骨架。O-GlcNAc在所有多细胞生物体中与蛋白质磷酸化一样丰富，并且与磷酸化具有动态相互作用。高血糖和高胰岛素血症诱导的O-GlcNAc升高阻断胰岛素信号传导并导致葡萄糖毒性。蛋白质的O-GlcNAc化对氧化应激和葡萄糖浓度都敏感。O-GlcNAc在不同蛋白质上以不同速率循环。将利用这些特性开发一种简单的免疫测定法，以定量位点特异性O-GlcNAc酰化，以便轻松评估患者血糖失调的程度和持续时间。计划书：在R21阶段，将使用两种互补的蛋白质组学方法（2D DIGE/MS-MS和BEMAD/MS-MS）来鉴定和定量样品中的O-GlcNAc位点，涵盖葡萄糖失调的一系列水平。该阶段的目的1使用STZ-大鼠模型将高血糖的幅度和时程与血液蛋白上特定位点的O-GlcNAc的变化相关联。在目标2中，我们将这些蛋白质组学方法应用于NIDDK和我们自己的“正常”，糖尿病前期和糖尿病受试者的良好表征，掩蔽，人类样本。R21的主要里程碑是鉴定了血液蛋白上的O-GlcNAc位点，这些位点对糖尿病状态显示出一致的反应范围。在R33阶段目标3中，将针对目标2中确定的未修饰和修饰位点制备位点特异性单克隆抗体。在目标4中，这些抗体将用于开发简单快速的定量免疫测定。 这些研究将利用一种普遍存在的，高度动态的蛋白质修饰，这是糖尿病病理生理异常的核心，以开发一种评估工具，该工具将比现有方法具有显着优势。该项目还代表了生物化学和医学系之间新的跨学科，跨部门的基础科学-临床研究合作。

DESCRIPTION (provided by applicant): The dynamic modification of nucleocytoplasmic proteins by the enzymatic attachment of O-linked N-acetylglucosamine to serine or threonine residues (O-GlcNAc) is now known to be a nutrient & stress sensor, regulating cellular signaling, transcription, proteasomal activity, and cytoskeleton. O-GlcNAc is as abundant as protein phosphorylation in all multi-cellular organisms, and has a dynamic interplay with phosphorylation. Hyperglycemia- and hyperinsulinemia-induced elevation of O-GlcNAc blocks insulin signaling and contributes to glucose toxicity. O-GlcNAcylation of proteins is sensitive to both oxidative stress and to glucose concentrations. O-GlcNAc cycles at different rates on different proteins. These properties will be exploited to develop a simple immunoassay to quantify site-specific O-GlcNAcylation in order to allow easy assessment of the extent and duration of glucose dysregulation in patients. Plan: In the R21 phase, two complementary proteomic approaches (2D DIGE/MS-MS & BEMAD/MS-MS) will be used to identify and quantify O-GlcNAc sites in samples covering a range levels of glucose dysregulation. Aim 1 of this phase uses the STZ-rat model to relate the amplitude and time course of hyperglycmia to changes in O-GlcNAc at specific sites on blood proteins. In Aim 2, we apply these proteomic methods to well characterized, masked, human samples from NIDDK and our own 'normal', pre-diabetic and diabetic subjects. The major milestone of the R21 is the identification of O-GlcNAc sites on blood proteins that display a consistent range of responsiveness to the diabetic state. In the R33 phase Aim 3, site specific monoclonal antibodies will be prepared to both the unmodified and the modified sites identified in Aim 2. In Aim 4, these antibodies will be used to develop simple and rapid quantitative immunoassays. These studies will exploit a ubiquitous, highly-dynamic protein modification that is central to the pathophysiologic abnormalities of diabetes, to develop an assessment tool that will have significant advantages over existing methods. The project also represents a new inter-disciplinary, inter-departmental basic science-clinical research collaboration between Biological Chemistry and The Department of Medicine.