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

项目摘要 O-GlcNAc转移酶（OGT）是一种人体必需的蛋白质，它将N-乙酰葡萄糖胺（GlcNAc）连接到Ser/Thr上， 细胞核和细胞质中的蛋白质残基。OGT表达和活性失调与 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。发展中 利用OGT作为靶点的方法需要了解其生理作用和机制， 它实现了它们。探索OGT的生理作用的研究严重依赖于敲低（KD）或敲低（KD）。 条件性敲除（KO）实验和所得表型已归因于O-GlcNAc的损失。 然而，OGT具有第二催化活性，并且还结合其不糖基化的细胞蛋白质;一些 因此，归因于O-GlcNAc损失的表型可能是由于其他活性的损失。此前 OGT的催化和非催化功能的重要性无法评估，因为替代方法 不存在具有变体的内源性OGT。我们现在已经建立了替代OGT的方法，并将分析 含有特异性改变的拷贝以解卷积OGT的生理作用的细胞。生长表型， 定量蛋白质组学和生物化学将用于解决几个问题。OGT的非催化性 支架活动是生理学的主要驱动力，与之相关的途径是什么？最短的OGT是多少 仍然支持细胞存活的结构，它糖基化和结合什么蛋白质？OGT如何 底物和结合配偶体与TPR结构域相互作用， 这个领域的？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， 筛选以鉴定对O-GlcNAc损失的遗传脆弱性。除了提高科学知识， 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 为指导OGT作为治疗靶点的方法奠定了基础。

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.