Regulation of IGF-1 and Insulin Signaling by O-GlcNAc Glycosylation

O-GlcNAc 糖基化对 IGF-1 和胰岛素信号传导的调节

基本信息

批准号：
8682801
负责人：
Lauren Elizabeth Ball
金额：
$ 36.51万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8682801
关键词：
Acetylglucosamine Address Alveolar Bone Loss Animal Model Animals Antibodies Apoptotic Bacterial Infections Binding Bone remodeling Calcified Calvaria Cell Line Cell Nucleus Cell Proliferation Cell model Cells Cellular Stress Chronic Coupling Cytoplasm Data Deposition Diabetes Mellitus Enzymes Excision Exhibits Genetic Polymorphism Glucose Goals Growth Factor Hormones Human Hyperglycemia In Vitro Infection Inflammatory Insulin Insulin Receptor Insulin-Like Growth Factor I Measures Mediating Methodology Modification Mus Mutate Natural regeneration Newly Diagnosed Nodule O-GlcNAc transferase Osteoblasts Osteoclasts Osteogenesis Osteoporosis Pathogenesis Pathway interactions Periodontal Diseases Periodontium Phosphorylation Post-Translational Protein Processing Post-Translational Regulation Predisposition Proteins Recruitment Activity Regulation Relative (related person)Research Role Signal Transduction Signaling Molecule Site Small Interfering RNA Specificity Staging Testing Tissues Tooth Loss Transfection Tyrosine Phosphorylation alveolar bone base bone cytokine diabetic diabetic patient glycosylation in vivo inhibitor/antagonist insulin signaling migration mineralization novel osteoclastogenesis overexpression peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase protein function receptor response src Homology Region 2 Domain stem stoichiometry therapeutic target

项目摘要

DESCRIPTION (provided by applicant): One third of all diabetic patients exhibit severe periodontal disease which is postulated to stem from an interplay of factors including increased susceptibility to infection, elevated pro-inflammatory cytokines, altered sensitivity to hormones, and hyperglycemia. The delayed regeneration of alveolar bone in the damaged periodontium reflects, in part, inadequate response to the anabolic actions of IGF-1 and insulin which may be down-regulated by cytokines and hyperglycemia. IGF-1 stimulation of osteoblasts, is anti-apoptotic and contributes to cell proliferation, differentiation, migration, matrix deposition and mineralization, and supports osteoclastogenesis. The specificity of the pleiotropic effects elicited by IGF-1R activation are mediated by the insulin receptor substrates, IRS-1 and IRS-2, which are critical to the formation of new bone and maintaining coupling between osteoblasts and osteoclasts. Signaling through the IRS proteins to PI3K/Akt and/or the Grb2/ERK pathways is mediated and modulated by posttranslational regulation. Perturbations in Tyr/Ser/Thr phosphorylation of IRS-1 occur in diabetes and osteoporosis. IRS-1 and IRS-2 are also glycosylated at Ser/Thr residues by N-acetylglucosamine (O-GlcNAc). This unique glycosylation is a reversible posttranslational modification proposed to regulate protein function in a manner analogous to phosphorylation. Using state-of-the-art tandem mass spectrometric approaches, we have identified multiple sites of O-GlcNAc modification on human IRS-1 and IRS-2 and developed methodology to measure the relative stoichiometry of this labile modification in a site-specific manner. The extent of protein O-GlcNAcylation changes rapidly in response to glucose, hormones, and cellular stress and it is implicated in altered insulin signaling and the glucose-induced complications associated with diabetes. In Aim 1, we propose to characterize the sites of O- GlcNAc modification of IRS-1 and IRS-2 in an osteoblast cell line and define the factors that modulate the extent of modification. We will investigate the interplay of phosphorylation and O-GlcNAc modification as these modifications may mutually exclude one another. The sites of O-GlcNAc modification of IRS-1 identified thus far are in close proximity to multiple SH2 domain binding motifs, thus in Aim 2 we will interrogate the effect of site-specific O-GlcNAc modification on interactions with the receptor and known binding partners and probe for novel protein interactions that may be induced by this modification. The role of the enzymes responsible for catalyzing the incorporation and removal of GlcNAc from proteins, O-GlcNAc transferase (OGT) and O- GlcNAcase (OGA), in modulating IGF-1 action in osteoblasts has not been addressed. Thus, in Aim 3, we will determine the impact of modulation of the expression of these enzymes on the cellular effects elicited by IGF-1 stimulation and whether altered O-GlcNAc modification of IRS-1 influences IGF-1 signaling in osteoblasts.

描述（由申请人提供）：三分之一的糖尿病患者表现出严重的牙周病，推测这种疾病源于多种因素的相互作用，包括感染易感性增加、促炎细胞因子升高、对激素敏感性改变和高血糖。受损牙周组织中牙槽骨再生的延迟部分反映了对 IGF-1 和胰岛素合成代谢作用的反应不足，而 IGF-1 和胰岛素可能会被细胞因子和高血糖下调。 IGF-1 刺激成骨细胞，具有抗凋亡作用，有助于细胞增殖、分化、迁移、基质沉积和矿化，并支持破骨细胞生成。 IGF-1R 激活引起的多效性效应的特异性是由胰岛素受体底物 IRS-1 和 IRS-2 介导的，它们对于新骨的形成以及维持成骨细胞和破骨细胞之间的偶联至关重要。通过 IRS 蛋白向 PI3K/Akt 和/或 Grb2/ERK 途径发出的信号由翻译后调节介导和调节。 IRS-1 的 Tyr/Ser/Thr 磷酸化扰动发生在糖尿病和骨质疏松症中。 IRS-1 和 IRS-2 的 Ser/Thr 残基也被 N-乙酰葡糖胺 (O-GlcNAc) 糖基化。这种独特的糖基化是一种可逆的翻译后修饰，旨在以类似于磷酸化的方式调节蛋白质功能。使用最先进的串联质谱方法，我们确定了人 IRS-1 和 IRS-2 上 O-GlcNAc 修饰的多个位点，并开发了以位点特异性方式测量这种不稳定修饰的相对化学计量的方法。蛋白质 O-GlcNAc 酰化程度会因葡萄糖、激素和细胞应激而迅速变化，并且与胰岛素信号传导的改变以及葡萄糖诱导的糖尿病相关并发症有关。在目标 1 中，我们建议表征成骨细胞系中 IRS-1 和 IRS-2 的 O-GlcNAc 修饰位点，并定义调节修饰程度的因素。我们将研究磷酸化和 O-GlcNAc 修饰的相互作用，因为这些修饰可能相互排斥。迄今为止鉴定的 IRS-1 的 O-GlcNAc 修饰位点与多个 SH2 结构域结合基序非常接近，因此在目标 2 中，我们将探讨位点特异性 O-GlcNAc 修饰对与受体相互作用和已知结合的影响伙伴并探索可能由这种修饰诱导的新蛋白质相互作用。负责催化 GlcNAc 从蛋白质中掺入和去除的酶、O-GlcNAc 转移酶 (OGT) 和 O-GlcNAcase (OGA) 在调节成骨细胞中 IGF-1 作用中的作用尚未得到解决。因此，在目标 3 中，我们将确定这些酶的表达调节对 IGF-1 刺激引起的细胞效应的影响，以及 IRS-1 的 O-GlcNAc 修饰改变是否会影响成骨细胞中的 IGF-1 信号传导。