Development of Site-specific O-GlcNAc Antibodies for Epigenetic Research

项目摘要 单一β-N-乙酰-D-氨基葡萄糖(O- GlcNAc)是一种常见的翻译后修饰，它是高度动态的，并随着细胞 刺激物。到目前为止，已经在大约1000种人类蛋白质上发现了这种类型的糖基化，并且是 被认为几乎和蛋白质磷酸化一样广泛和丰富。事实上，O-GlcNAc经常 与蛋白质磷酸化竞争，这两个修饰在调节中有广泛的串扰 信号、转录以及癌基因和肿瘤抑制因子的功能。修改似乎是为了 在癌症、阿尔茨海默病和糖尿病等关键病理生理疾病中发挥重要作用。 许多最先鉴定出携带这种修饰的蛋白质是转录因子，它已经成为 最近几年的研究表明，O-GlcNAc在染色质重塑和基因表达中起着重要作用。 这项建议的重点是开发部位特异性抗体，作为工具来阐明 O-GlcNAc在表观遗传学中的作用在之前的第一阶段授权中，我们专注于评估合成 免疫原，产生针对O-GlcNAc修饰的四个位点的多克隆抗体(PAb) 核心组蛋白(组蛋白2A、2B、3和4)，并鉴定这些抗体。我们每一次都很成功 这是前一个项目的目标，这导致了这个第二阶段的提案。在这里，我们建议使用我们的专有 免疫策略，大幅扩大我们的位点特异性O-GlcNAc抗体库，以包括 目前已知的以这种方式修饰的大多数蛋白质也参与了基因表达。 因此，如果我们成功，研究人员将有机会获得广泛开发的特定部位抗体 表观遗传学研究，因此我们认为这项研究将对表观遗传学研究产生立竿见影的影响 并可能对疾病研究产生深远的影响。

Project Summary O-glycosylation of nuclear and cytoplasmic proteins by a single β-N-acetyl-D-glucosamine moiety (O- GlcNAc) is a common post-translational modification that is highly dynamic and fluctuates in response to cellular stimuli. This type of glycosylation has been found on approximately a thousand human proteins to date, and is thought to be nearly as wide-spread and abundant as protein phosphorylation. In fact, O-GlcNAc often competes with protein phosphorylation, and these two modifications have extensive crosstalk in the regulation of signaling, transcription, and the functions of oncogenes and tumor suppressors. The modification appears to play a major role in key pathophysiological conditions including cancer, Alzheimer’s disease, and diabetes. Many of the first proteins identified carrying this modification were transcription factors, and it has become clear in the last several years that O-GlcNAc plays a major role in chromatin remodeling and gene expression. The focus of this proposal is to develop site-specific antibodies that can be used as tools in the elucidation of the role that O-GlcNAc plays in epigenetics. In the predecessor Phase I grant we focused on evaluating synthetic immunogens, the production of polyclonal antibodies (PAbs) to five sites of O-GlcNAc modification on the four core histones (Histone 2A, 2B, 3, and 4), and characterizing these antibodies. We were successful with each Aim of this previous project, which leads to this Phase II proposal. Here, we propose to utilize our proprietary immunization strategy to significantly expand our repertoire of site-specific O-GlcNAc antibodies to include the majority of proteins currently known to be modified in this manner that are also involved in gene expression. Consequently, if we are successful, researchers will have access to a wide-range of site-specific Abs developed for epigenetic research, and thus we feel that this study will have an immediate impact on epigenetic research and could have far reaching implications in disease research.