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

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，这被认为是由多种因素的相互作用引起的，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症。受损牙周组织中牙槽骨的延迟再生部分反映了对IGF-1和胰岛素的合成代谢作用的反应不足，IGF-1和胰岛素可能被细胞因子和高血糖下调。IGF-1刺激成骨细胞，是抗凋亡的，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。由IGF-1 R激活引起的多效性效应的特异性由胰岛素受体底物IRS-1和IRS-2介导，这对新骨的形成和维持成骨细胞与破骨细胞之间的偶联至关重要。通过IRS蛋白到PI 3 K/Akt和/或Grb 2/ERK途径的信号传导由翻译后调节介导和调节。IRS-1的Tyr/Ser/Thr磷酸化的扰动发生在糖尿病和骨质疏松症中。IRS-1和IRS-2也在Ser/Thr残基处被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，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，我们已经确定了人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化的程度响应于葡萄糖、激素和细胞应激而迅速变化，并且其涉及改变的胰岛素信号传导和与糖尿病相关的葡萄糖诱导的并发症。在目的1中，我们提出表征成骨细胞系中IRS-1和IRS-2的O-GlcNAc修饰的位点，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。迄今为止确定的IRS-1的O-GlcNAc修饰位点与多个SH 2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修饰对与受体和已知结合伙伴相互作用的影响，并探测可能由这种修饰诱导的新型蛋白质相互作用。负责催化GlcNAc从蛋白质掺入和去除的酶O-GlcNAc转移酶（OGT）和O-GlcNAc酶（OGA）在成骨细胞中调节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的调节对IGF-1刺激引起的细胞效应的影响，以及IRS-1的改变的O-GlcNAc修饰是否影响成骨细胞中的IGF-1信号传导。 公共卫生相关性：随着美国每年新诊断的糖尿病病例数量的预计增加，了解对糖尿病及其并发症的发病机理至关重要的关键分子的调节是至关重要的。三分之一的糖尿病患者表现出严重的牙周病，其原因是慢性细菌感染，但随着与糖尿病状态相关的潜在变化，对激素和细胞因子的反应发生改变，最终导致牙槽骨退化和牙齿脱落。这项研究将提供新的信息有关的两个蛋白质的正常骨形成的调节，是已知的干扰在糖尿病与阐明治疗目标，以促进再生的牙槽骨在糖尿病相关的牙周病的最终目标。

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. PUBLIC HEALTH RELEVANCE: With the predicted escalation in the number of newly diagnosed cases of diabetes annually in the U.S., understanding the regulation of key molecules critical to the pathogenesis of diabetes and its complications is essential. One-third of diabetic patients exhibit severe periodontal disease, the cause of which is a chronic bacterial infection, but with the underlying changes associated with the diabetic state, the stage is set for an altered response to hormones and cytokines culminating in the degradation of alveolar bone and tooth loss. This research will provide novel information concerning the regulation of two proteins crucial for normal bone formation that are known to be perturbed in diabetes with the ultimate goal of elucidating therapeutic targets to facilitate regeneration of alveolar bone in diabetes-associated periodontal disease.