Defining OGT's Essential Functions to Guide Therapeutic Approaches

项目总结 O-GlcNAc转移酶(OGT)是一种人体必需的蛋白质，将N-乙酰氨基葡萄糖(GlcNAc)连接到丝氨酸/苏氨酸上 细胞核和细胞质中的蛋白质残留物。异常的OGT表达和活性被联系在一起 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 开发OGT作为靶点的方法需要了解它的生理作用和机制 而它实现了这些目标。探索OGT的生理作用的研究在很大程度上依赖于基因敲除(KD)或 条件性基因敲除(KO)实验和由此产生的表型被归因于O-GlcNAc的丢失。 然而，OGT具有第二种催化活性，它还结合了它不糖化的细胞蛋白质；一些 因此，O-GlcNAc缺失的表型可能是由于其他活动的缺失所致。此前， 无法评估OGT的催化和非催化功能的重要性，因为替代 不存在内源性OGT及其变异体。我们现在已经建立了取代OGT的方法，并将分析 含有特定改变的拷贝的细胞，以解卷OGT的生理作用。生长表型， 定量蛋白质组学和生物化学将被用来解决几个问题。OGT是否是非催化的 脚手架活动是生理学的主要驱动力，有哪些途径与此有关？最短的OGT是什么 构建仍然支持细胞生存的结构，以及它糖基化和结合哪些蛋白质？OGT是如何 底物和结合伙伴与TPR结构域相互作用，是否有机会针对特定的 此域的区域？我们还将在化学基因组学中使用我们最近开发的一种小分子抑制剂 筛查以确定O-GlcNAc缺失的遗传易感性。除了促进关于 是哺乳动物生物学中最基本的重要蛋白质之一，本提案中的工作将提供 指导将OGT作为治疗靶点的方法的基础。

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.