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

项目摘要 通过单个β-N-乙酰基-D-葡糖胺部分（O- GlcNAc）是一种常见的翻译后修饰，其是高度动态的，并且响应于细胞内信号而波动。 刺激。迄今为止，在大约一千种人类蛋白质上发现了这种类型的糖基化， 被认为几乎和蛋白质磷酸化一样广泛和丰富。事实上，O-GlcNAc通常 与蛋白质磷酸化竞争，这两种修饰在调节中具有广泛的串扰 信号传导、转录以及癌基因和肿瘤抑制因子的功能。修改似乎 在包括癌症、阿尔茨海默病和糖尿病在内的关键病理生理条件中发挥重要作用。 许多第一批被鉴定出的携带这种修饰的蛋白质是转录因子，它已经成为 在过去的几年中，O-GlcNAc在染色质重塑和基因表达中起着重要作用。 该建议的重点是开发位点特异性抗体，其可用作阐明 O-GlcNAc在表观遗传学中的作用。在前一期赠款中，我们专注于评估合成 免疫原，生产多克隆抗体（PAbs）的五个位点的O-GlcNAc修饰的四个 核心组蛋白（组蛋白2A、2B、3和4），并表征这些抗体。我们每一次都很成功 这是前一个项目的目的，这导致了第二阶段的建议。在这里，我们建议利用我们的专有技术 免疫策略，以显着扩大我们的位点特异性O-GlcNAc抗体的库，包括 目前已知的大多数蛋白质以这种方式被修饰，它们也参与基因表达。 因此，如果我们成功了，研究人员将有机会获得广泛的网站特定的抗体开发 因此，我们认为这项研究将对表观遗传学研究产生直接影响 并且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.