Defining OGT's Essential Functions to Guide Therapeutic Approaches - EQUIPMENT SUPPLEMENT

定义 OGT 的基本功能以指导治疗方法 - 设备补充

• 批准号: 10386477
• 负责人: Suzanne Walker
• 金额: $ 7万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386477
• 关键词: Address; Binding; Biochemistry; Biology; Cardiometabolic Disease; Cell Nucleus; Cell Survival; Cells; Complications of Diabetes Mellitus; Cytoplasm; Drug Targeting; Foundations; Genetic Predisposition to Disease; Growth; Human; Insulin Resistance; Knowledge; Link; Malignant Neoplasms; Mammals; Methods; O-GlcNAc transferase; Pathway interactions; Phenotype; Physiological; Physiology; Proteins; Proteomics; Role; Scientific Advances and Accomplishments; Supporting Cell; Technology; Therapeutic; Variant; Work; cancer therapy; conditional knockout; experimental study; knock-down; scaffold; small molecule inhibitor; therapeutic target; tool

PROJECT SUMMARY O-GlcNAc transferase (OGT), an essential human protein, attaches N-acetylglucosamine (GlcNAc) to Ser/Thr residues of proteins in the nucleus and cytoplasm. Dysregulated OGT expression and activity have been linked to insulin resistance, diabetic complications, and cancer, making OGT a possible drug target. Developing approaches to exploit OGT as a target requires understanding its physiological roles and the mechanisms by which it achieves them. Studies to probe OGT’s physiological roles have relied heavily on knockdown (KD) or conditional knockout (KO) experiments and the resulting phenotypes have been attributed to loss of O-GlcNAc. However, OGT has a second catalytic activity and also binds cellular proteins that it does not glycosylate; some phenotypes attributed to O-GlcNAc loss may therefore be due to loss of other activities. Previously, the importance of OGT’s catalytic and noncatalytic functions could not be assessed because methods to replace endogenous OGT with variants did not exist. We have now established methods to replace OGT and will analyze cells containing specifically altered copies to deconvolute OGT’s physiological roles. Growth phenotypes, quantitative proteomics, and biochemistry will be used to address several questions. Is OGT’s noncatalytic scaffolding activity a major driver of physiology and what pathways are linked to it? What is the shortest OGT construct that still supports cell survival and what proteins does it glycosylate and bind? How do OGT’s substrates and binding partners interact with the TPR domain and are there opportunities to target specific regions of this domain? We will also use a small molecule inhibitor we recently developed in a chemogenomics screen to identify genetic vulnerabilities to loss of O-GlcNAc. In addition to advancing scientific knowledge about one of the most fundamentally important proteins in mammalian biology, the work in this proposal will provide the foundation to guide approaches to exploit OGT as a therapeutic target.

项目概要 O-GlcNAc 转移酶 (OGT) 是一种必需的人类蛋白质，可将 N-乙酰氨基葡萄糖 (GlcNAc) 连接到 Ser/Thr 细胞核和细胞质中蛋白质残基的失调与 OGT 表达和活性有关。 胰岛素抵抗、糖尿病并发症和癌症，使 OGT 成为可能的药物靶点。 利用 OGT 作为靶点的方法需要了解其生理作用和机制 探究 OGT 生理作用的研究在很大程度上依赖于敲低 (KD) 或 条件敲除（KO）实验和由此产生的表型被归因于 O-GlcNAc 的损失。 然而，OGT 具有第二种催化活性，并且还可以结合一些不被糖基化的细胞蛋白； 因此，归因于 O-GlcNAc 损失的表型可能是由于其他活性的损失。 OGT 催化和非催化功能的重要性无法评估，因为替代方法 不存在内源性 OGT 变异，我们现在已经建立了替代 OGT 的方法，并将进行分析。 含有专门改变的拷贝以解开 OGT 的生理作用的细胞， 蛋白质组学和生物化学将用于解决 OGT 的非催化性几个定量问题。 支架活动是生理学的主要驱动因素，以及与之相关的途径是什么？ 仍然支持细胞存活的构建体以及它如何糖基化和结合哪些蛋白质？ 底物和结合伴侣与 TPR 结构域相互作用，是否有机会靶向特定的 我们还将使用我们最近在化学基因组学中开发的小分子抑制剂 除了提高有关 O-GlcNAc 的科学知识之外，还进行筛查以确定 O-GlcNAc 损失的遗传脆弱性。 作为哺乳动物生物学中最重要的蛋白质之一，本提案中的工作将提供 指导利用 OGT 作为治疗目标的方法的基础。