Structure, Function and Inhibition of Human O-GlcNAc Transferase

 描述（由申请人提供）：O-GlcNAc转移酶（OGT）存在于所有后生动物中，是哺乳动物胚胎发育所必需的，并且在整个生命周期中持续需要增殖细胞的活力。尽管OGT在生物学中的重要性是无可争议的，但其功能仍然知之甚少。它具有三种不同的生物化学活性：1）阿萨糖基转移酶，将N-乙酰葡糖胺（GlcNAc）附着到多种细胞质和核蛋白上，从而影响它们的稳定性、定位和生物化学功能 响应变化的细胞条件; 2）它在HCF-1成熟中作为蛋白酶发挥作用，HCF-1是细胞周期进程所需的必需的多结构域转录共调节因子;和3）它作为与几种多蛋白复合物的组分相互作用的支架蛋白。OGT与包括癌症和糖尿病在内的涉及葡萄糖摄取和代谢失调的疾病有关，并且是一种建议的治疗靶点。本文提出的研究结合了化学合成，生物化学和细胞生物学，以更好地了解OGT的不同活性，这对于评估其作为治疗靶点的潜力至关重要。目标1的重点是基于上一个资助期发现的先导化合物开发细胞渗透性小分子抑制剂。这些抑制剂将有助于研究OGT的细胞活性，并对研究其在细胞信号传导中的作用特别重要。目的二是利用蛋白质微阵列研究OGT的结构特征，这些结构特征对底物选择很重要。这些研究可能揭示OGT TPR结构域的不同部分参与选择不同的底物子集，这一结果将对途径选择性OGT抑制剂产生影响。目标3集中于测试我们提出的OGT如何切割HCF-1的机制。这些研究是有必要的，因为机制，如发现OGT使用相同的活性位点进行蛋白质O-GlcNAc酰化和蛋白质水解，是前所未有的生物学。最后，目标4的重点是建立一个遗传系统，以取代野生型OGT与OGT变体缺乏特定的生物化学活性，使我们可以解决以下基本问题：为什么是OGT需要增殖的哺乳动物细胞的生存？是否需要HCF-1裂解？是否需要OGT的脚手架功能？或者O-GlcNAc化活性是必需的，如果是，什么靶点是最重要的？一个强大的遗传系统来研究OGT变体将使我们能够将生化研究的结果与细胞表型联系起来，从而更好地理解OGT生物学。

 DESCRIPTION (provided by applicant): O-GlcNAc transferase (OGT), found in all metazoans, is essential for embryonic development in mammals and continues to be required throughout life for the viability of proliferating cells. Although the importance of OGT in biology is not disputed its functions remain poorly understood. It has three distinct biochemical activities: 1) it acts asa glycosyltransferase, attaching N-acetylglucosamine (GlcNAc) to a wide variety of cytoplasmic and nuclear proteins and thereby affecting their stability, localization, and biochemical functions in response to changing cellular conditions; 2) it functions as a protease in the maturation of HCF-1, an essential multi-domain transcriptional co-regulator required for cell cycle progression; and 3) it serves as a scaffolding protein that interacts with components of several multi-protein complexes. OGT has been implicated in diseases involving dysregulated glucose uptake and metabolism, including cancer and diabetes, and it is a proposed therapeutic target. The research proposed here combines chemical synthesis, biochemistry, and cell biology to gain a better understanding of OGT's different activities, which is critical for assessing its potential a a therapeutic target. Aim 1 focuses on the development of cell permeable small molecule inhibitors based on a lead discovered in the previous funding period. These inhibitors will be useful for investigating OGT's cellular activities and are particularly important for studies of it roles in cell signaling. Aim 2 focuses on using protein microarrays to investigate the structural features of OGT that are important for substrate selection. These studies may reveal that different parts of the OGT TPR domain are involved in selecting different subsets of substrates, a result that would have implications for pathway-selective OGT inhibitors. Aim 3 focuses on testing our proposed mechanism for how OGT cleaves HCF-1. These studies are warranted because the mechanism, like the discovery that OGT uses the same active site for both protein O-GlcNAcylation and proteolysis, is unprecedented in biology. Finally, Aim 4 focuses on establishing a genetic system to replace wildtype OGT with OGT variants deficient in a particular biochemical activity so that we can address the following fundamental questions: Why is OGT required for survival of proliferating mammalian cells? Is HCF-1 cleavage required? Is OGT's scaffolding function required? Or is O-GlcNAcylation activity necessary, and, if so, what targets are most important? A robust genetic system to investigate OGT variants will allow us to link findings from biochemical studies to cellular phenotypes, leading to a better understanding of OGT biology.