Chemical Approaches to Understand the Roles of O-GlcNAc Glycosylation in Biology

描述（由申请人提供）：碳水化合物包括生物活性分子的最大和最多样化的集合之一。然而，相对于其他生物分子，如核酸和蛋白质，碳水化合物仍然相对未开发，其结构-功能关系仍然知之甚少。该计划的主要目标是开发化学方法，以促进对碳水化合物在生物学和疾病中的作用的基本理解。在上一个授权期，我们开发了快速、灵敏地检测和研究O-连接的<$-D-N-乙酰氨基葡萄糖（O-GlcNAc）糖基化的化学方法。O-GlcNAc是一种丰富的、必需的翻译后修饰，其正在成为许多生理功能的关键调节剂，范围从表观遗传和转录基因调节到胰岛素信号传导、癌细胞代谢和神经变性。我们的化学方法使第一个蛋白质组范围内的分析的修改和调查的动力学，化学计量，和位点特异性功能的O-GlcNAc。在即将到来的拨款期内，我们的目标是解决下一组关键障碍， 外地在目标1中，我们将开发新的方法来了解O-GlcNAc转移酶（OGT）酶的复杂调控。我们的研究将确定OGT的蛋白质相互作用和底物网络，OGT内的特定结构域如何对这些网络做出贡献，以及细胞刺激如何动态地改变网络。这些研究将解决一个单一的酶如何调节这么多不同的生物过程的核心问题。在目标2中，我们将通过开发用于均匀O-GlcNAc酰化蛋白质的半合成的一般化学方法来解决对O-GlcNAc修饰的蛋白质的详细结构和生化研究的迫切需要。在目标3中，我们将继续我们令人兴奋的发现，即糖基化磷酸果糖激酶1（PFK 1）可能代表一种新的抗癌靶点，通过开发调节PFK 1活性的化合物并测试其对癌症代谢和生长的影响。在目的4中，我们将通过研究O-GlcNAc在PI 3 K-Akt-mTOR通路中的作用来研究其在调节细胞代谢中的更广泛作用，PI 3 K-Akt-mTOR通路是许多癌症中经常失调的通路。了解O-GlcNAc化如何调节mTOR信号传导可能提供一种新的方法来调节这一关键途径，并导致发现新的治疗干预策略。总的来说，该项目将为O-GlcNAc的功能提供重要的新见解，并导致新方法，新假设和生物学发现，这将推动该领域的发展。此外，这项工作有望揭示新的潜在治疗靶点和/或方法。最后，所提出的实验的一个独特的方面是化学和生物学的无缝集成，我们认为这是一个强大的组合，用于获得碳水化合物的结构-功能关系的基本见解，并推进化学生物学，糖生物学和癌症生物学的前沿。

DESCRIPTION (provided by applicant): Carbohydrates comprise one of the largest and most diverse collections of biologically-active molecules. However, relative to other biomolecules such as nucleic acids and proteins, carbohydrates remain relatively unexplored, and their structure-function relationships are still poorly understood. The broad objective of this program is to develop chemical approaches to advance a fundamental understanding of the roles of carbohydrates in biology and disease. In the last granting period, we developed chemical methods for the rapid, sensitive detection and study of O-linked ¿-D-N-acetylglucosamine (O-GlcNAc) glycosylation. O-GlcNAc is an abundant, essential post-translational modification that is emerging as a key regulator of many physiological functions, ranging from epigenetic and transcriptional gene regulation to insulin signaling, cancer cell metabolism, and neurodegeneration. Our chemical methods enabled the first proteome-wide analyses of the modification and investigations into the dynamics, stoichiometry, and site-specific functions of O-GlcNAc. In the coming granting period, our goal is to tackle the next set of critical barriers in the field. In Aim 1, we will develop new methods to understand the complex regulation of the O-GlcNAc transferase (OGT) enzyme. Our studies will determine the protein interaction and substrate networks of OGT, how specific structural domains within OGT contribute to those networks, and how cellular stimuli dynamically alter the networks. These studies will address the central question of how a single enzyme regulates so many diverse biological processes. In Aim 2, we will address the critical need for detailed structural and biochemical studies of O-GlcNAc-modified proteins by developing a general chemical method for the semi-synthesis of homogeneously O-GlcNAcylated proteins. In Aim 3, we will follow up on our exciting discovery that glycosylated phosphofructokinase 1 (PFK1) may represent a novel anti-cancer target by developing compounds that modulate PFK1 activity and testing their effects on cancer metabolism and growth. In Aim 4, we will investigate the broader roles of O-GlcNAc in regulating cellular metabolism by studying its role in the PI3K-Akt-mTOR pathway, a pathway frequently deregulated in many cancers. Understanding how O-GlcNAcylation regulates mTOR signaling may provide a novel approach to modulate this critical pathway and lead to the discovery of new strategies for therapeutic intervention. Overall, this project will provide essential new insights into the functions of O-GlcNAc and lead to new methods, novel hypotheses, and biological discoveries that will drive the field forward. In addition, the work is expected to reveal new potential therapeutic targets and/or approaches. Finally, a distinctive aspect of the proposed experiments is the seamless integration of chemistry and biology, which we believe is a powerful combination for obtaining fundamental insights into the structure-function relationships of carbohydrates and for advancing the frontiers of chemical biology, glycobiology, and cancer biology.