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

用于表观遗传学研究的位点特异性 O-GlcNAc 抗体的开发

• 批准号: 9347335
• 负责人: Marla Popov
• 金额: $ 35万
• 依托单位: GLYCOSCIENTIFIC, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9347335
• 关键词: Affinity; Affinity Chromatography; Alzheimer' s Disease; Amino Acid Sequence; Antibodies; Autoantigens; Binding; Biology; Cell physiology; Cells; Cellular biology; ChIP-seq; Collection; Complex; Cytoplasmic Protein; Detection; Development; Diabetes Mellitus; Disease; Enzyme-Linked Immunosorbent Assay; Enzymes; Epigenetic Process; Epitopes; Gene Expression; Gene Expression Regulation; Genetic Transcription; Glucosamine; Glycopeptides; Grant; Histones; Human; Hybridomas; Immune system; Immunization; Immunoprecipitation; Link; Malignant Neoplasms; Market Research; Modification; Monoclonal Antibodies; Nuclear; Nuclear Proteins; Oryctolagus cuniculus; Phase; Phospho-Specific Antibodies; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Polysaccharides; Post-Translational Protein Processing; Process; Production; Property; Protein-Carbohydrate Interaction; Proteins; Protocols documentation; Reagent; Regulation; Research; Research Personnel; Resources; Role; Scientist; Signal Transduction; Site; Specificity; Stimulus; Synthetic Vaccines; Transfer Factor; Tumor Suppressor Genes; UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; United States National Academy of Sciences; Western Blotting; analytical tool; chromatin remodeling; experience; glycosylation; human disease; monoclonal antibody production; novel; novel strategies; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; polyclonal antibody; preference; response; sugar; tool; transcription factor

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.

项目概要 单个 β-N-乙酰基-D-葡萄糖胺部分对核蛋白和细胞质蛋白进行 O-糖基化 (O- GlcNAc）是一种常见的翻译后修饰，它具有高度动态性，并且会响应细胞的变化而波动。 迄今为止，已在大约一千种人类蛋白质中发现了这种类型的糖基化。 人们认为 O-GlcNAc 几乎与蛋白质磷酸化一样广泛和丰富。 与蛋白质磷酸化竞争，这两种修饰在调控中存在广泛的串扰 这种修饰似乎与信号传导、转录以及癌基因和肿瘤抑制因子的功能有关。 在癌症、阿尔茨海默病和糖尿病等关键病理生理状况中发挥着重要作用。 许多首先鉴定出带有这种修饰的蛋白质是转录因子，并且它已成为 在过去的几年中，人们清楚地认识到 O-GlcNAc 在染色质重塑和基因表达中发挥着重要作用。 该提案的重点是开发位点特异性抗体，可用作阐明 O-GlcNAc 在表观遗传学中的作用在评估前一期资助时我们重点关注合成。 免疫原，对四个 O-GlcNAc 修饰的五个位点产生多克隆抗体 (PAb) 核心组蛋白（组蛋白 2A、2B、3 和 4），并对这些抗体进行了表征，我们对每种抗体都取得了成功。 上一个项目的目标导致了第二阶段的提案，在这里，我们建议利用我们的专有技术。 免疫策略显着扩展我们的位点特异性 O-GlcNAc 抗体库，包括 目前已知的大多数蛋白质都以这种方式修饰，并且也参与基因表达。 经过测试，如果我们成功，研究人员将能够获得广泛开发的特定位点抗体 对于表观遗传学研究，因此我们认为这项研究将对表观遗传学研究产生直接影响 并可能对疾病研究产生深远的影响。