Structure, Function and Inhibition of Human O-GlcNAc Transferase

 描述(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，并且在整个生命过程中仍然是增殖细胞生存所必需的。尽管OGT在生物学中的重要性是毋庸置疑的，但它的功能仍然知之甚少。它有三种不同的生化活性：1)作为糖基转移酶，将N-乙酰氨基葡萄糖(GlcNAc)附着到各种细胞质和核蛋白上，从而影响它们的稳定性、定位和生化功能 2)它在hcf-1的成熟过程中作为一种蛋白酶发挥作用，hcf-1是细胞周期进程所必需的一个重要的多结构域转录辅助调节因子；3)它作为一种支架蛋白与几种多蛋白复合体的成分相互作用。OGT与涉及葡萄糖摄取和代谢失调的疾病有关，包括癌症和糖尿病，它是一个被提议的治疗靶点。这里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侧重于在前一资金时期发现的铅的基础上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，对于研究OGT在细胞信号转导中的作用尤为重要。目的2利用蛋白质芯片研究OGT的结构特征，这些结构特征对底物的选择具有重要意义。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亚集，这一结果将对途径选择性OGT抑制剂产生影响。目标3专注于测试我们提出的OGT如何切割HCF-1的机制。这些研究是有必要的，因为这种机制在生物学上是史无前例的，就像发现OGT对蛋白质O-GlcN酰化和蛋白质分解使用相同的活性部位一样。最后，目标4侧重于建立一个遗传系统，用特定生化活性缺失的OGT变体取代野生型OGT，这样我们就可以解决以下基本问题：为什么OGT是增殖中的哺乳动物细胞生存所必需的？是否需要对hcf-1进行切割？是否需要OGT的脚手架功能？或者O-GlcN酰化活性是必要的，如果是的话，什么目标是最重要的？研究OGT变异的强大遗传系统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.