Beyond the Active Site: Structure Informed Novel Regulatory Mechanisms and Functional Modulation of O-GlcNAc Transferase

摘要 人体必需的O-GlcNAc转移酶(OGT)催化一种独特的细胞内蛋白 糖基化称为O-GlcN酰化。作为对营养水平和压力的反应，OGT动态调节 多种生理和病理过程，包括癌细胞和胰岛素的“华宝效应” 糖尿病的抵抗力。以往对OGT活性部位的研究在其上有了根本性的发现 催化机理和底物相互作用。然而，OGT如何调节蛋白质和位点特异性的O- GlcN酰化作用仍不清楚。这是由于许多挑战，包括：1)OGT糖基化数千 在O-GlcNAc修饰位点附近没有保守序列基序的蛋白质中，2)大多数O- GlcN酰化位点位于固有无序区(IDR)，3)OGT通常与蛋白质结合 低/中等亲和力，以及4)缺乏OGT-蛋白质复合体结构。在我们的上一个资助期，我们已经做出了 通过开发一套允许我们审问OGT的新型化学探针，在这些领域取得了很大进展 具有低/中等亲和力的特定相互作用，用于结构、蛋白质组和生物化学表征。这 该提案旨在进一步在概念和技术上突破，以解决这些长期存在的问题 挑战。人们期望对OGT如何与其他蛋白质相互作用有更好的了解，特别是 通过OGT催化位点以外的区域，将是理解OGT功能调控的关键 在蛋白质和特定部位的水平上，填补了数十年来对 OGT的营养传感和其他调节作用，并将支持专门调节OGT的需要 用于生物医学应用的功能。

Abstract The essential human enzyme O-GlcNAc transferase (OGT) catalyzes a unique type of intracellular protein glycosylation called O-GlcNAcylation. In response to nutrient levels and stress, OGT dynamically regulates a variety of physiological and pathological processes including the “Warburg effect” in cancer cells and insulin resistance in diabetes. Previous studies on the OGT active site have made fundamental discoveries on its catalytic mechanism and substrate interactions. However, how OGT regulates protein- and site-specific O- GlcNAcylation remains unclear. This is due to a number of challenges including: 1) OGT glycosylates thousands of proteins without a conserved sequence motif near the O-GlcNAc modification site, 2) a majority of O- GlcNAcylation sites are found on intrinsically disordered regions (IDRs), 3) OGT typically binds proteins with low/moderate affinity, and 4) a lack of OGT-protein complex structures. In our last funding period, we have made strides in these areas through development of a suite of novel chemical probes that allow us to interrogate OGT specific interactions with low/moderate affinity for structural, proteomic, and biochemical characterizations. This proposal aims to make further conceptual and technical breakthroughs toward addressing these longstanding challenges. It is expected that a better understanding of how OGT interacts with other proteins, particularly through the regions beyond the OGT catalytic site, will be essential for understanding OGT’s functional regulation at protein- and site-specific levels, filling major knowledge gaps between decades of biological observations of OGT’s nutrient sensing and other regulatory roles, and will support the need to specifically modulate OGT functions for biomedical applications.