Chemical tools for studying carbohydrates and carbohydrate processing enzymes

研究碳水化合物和碳水化合物加工酶的化学工具

• 批准号: RGPIN-2020-06466
• 负责人: Vocadlo, David
• 金额: $ 7.65万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748143
• 关键词: Chemical; tools; studying; carbohydrates; carbohydrate

Glycoconjugates are a ubiquitous class of biomolecules found in all kingdoms of life. Relative to other areas, however, advances in studying glycoconjugates have been hampered by a shortage of chemical tools and technologies to facilitate their investigation. The wide availability of robust chemical tools for studying proteins, nucleic acids, and widely recognized protein modifications such as phosphorylation, serves as an instructive foil to highlight the issue. A major thrust of our research program is directed toward developing chemical biology tools to fill this gap and thereby facilitate research into understanding various glycoconjugates and the enzymes that build them up (glycosyltransferases) and those that break them down (glycosidases). Here we outline a principally chemistry-based program of research focused on creation of enabling chemical biology tools. First, chemical kinetic studies of enzymes in vitro using standard chromogenic or fluorogenic substrates have provided critically important insights into how glycoside hydrolases act on their substrates. Ultimately, however, these enzymes function within the complex environment of the cell in the presence of metabolites and protein partners. Accordingly, we will advance our pioneering studies to synthesize bright fluorescence resonance energy transfer (FRET) quenched substrates for a suite of glycosidases. These probes will be usable in various formats to quantitatively measure the activities of functionally related human enzymes within cells in a multiplexed manner. Second, live cell enzymology is a frontier area having the goal of analyzing the activity of enzymes directly within their native milieu of living cells. This emerging field has, however, been hindered by a series of technical challenges. Here we aim to develop ratiometric "caged" substrates for two glycosidases and use these to establish the first detailed enzyme kinetic study in live cells by microscopy. This work will set the stage to understand how endogenous cellular factors influence enzyme function within cells. Third, we aim to chemically synthesize "caged" inhibitors of two related glycosidases and glycosyltransferases. Once these initially inactive caged inhibitors are irradiated with light the photoprotecting group is lost and the potent inhibitor is released. Such tools will permit spatial and temporal control over these important enzymes in cells and tissues. Together, progress toward these three aims should provide improved tools for observing and controlling glycosidases and glycosyltransferases. These new tools for use within cells should accelerate biological research into the regulation and functional importance of these enzymes and glycoconjugates. Many of the principles applied here should be applicable to other carbohydrate processing enzymes and, indeed, more broadly to other superfamilies of enzymes.

糖缀合物是在生活的所有王国中发现的无处不在的生物分子。然而，相对于其他领域，研究糖缀合物的进展受到化学工具和技术的短缺的阻碍，无法促进其研究。可用于研究蛋白质，核酸以及广泛认识的蛋白质修饰（例如磷酸化）的广泛可用性，是一种指导性的箔，以突出该问题。我们的研究计划的一个重大作用是针对开发化学生物学工具来填补这一空白的，从而促进了理解各种糖缀合物和构建它们的酶（糖基转移酶），以及那些分解它们的酶（糖苷酶）。在这里，我们概述了一个主要基于化学的研究计划，该计划着重于创建化学生物学工具。首先，使用标准的发色或荧光底物对酶进行化学动力学研究，对糖苷水解酶如何在其底物上作用非常重要。然而，最终，这些酶在代谢物和蛋白质伴侣的存在下在细胞的复杂环境中起作用。因此，我们将提高开拓性研究，以合成一组糖苷酶套件的明亮荧光共振能量转移（FRET）淬灭底物。这些探针将以各种格式可用，以定量测量细胞中与功能相关的人类酶的活性。其次，活细胞酶学是一个前沿区域，其目的是直接在活细胞的天然环境中分析酶的活性。但是，这一新兴领域受到一系列技术挑战的阻碍。在这里，我们旨在为两种糖苷酶开发比率的“笼”底物，并使用它们通过显微镜在活细胞中建立第一个详细的酶动力学研究。这项工作将奠定阶段，以了解内源性细胞因子如何影响细胞内的酶功能。第三，我们旨在化学合成两种相关糖苷酶和糖基转移酶的“笼”抑制剂。一旦这些最初无活跃的笼抑制剂被光照射，光保护组就会丢失，并释放了有效的抑制剂。这样的工具将允许对细胞和组织中这些重要酶的空间和时间控制。共同朝着这三个目标方面的进展应提供改进的工具，用于观察和控制糖苷酶和糖基转移酶。这些新的在细胞中使用的新工具应加快这些酶和糖缀合物的调节和功能重要性的生物学研究。这里应用的许多原理应适用于其他碳水化合物加工酶，实际上更广泛地用于其他酶的超级家族。