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.

糖缀合物是一种普遍存在于所有生命领域的生物分子。然而，相对于其他领域，糖缀合物的研究进展受到化学工具和技术的缺乏的阻碍。用于研究蛋白质、核酸和广泛认可的蛋白质修饰（如磷酸化）的强大化学工具的广泛可用性，有助于突出这一问题。我们研究项目的一个主要目标是开发化学生物学工具来填补这一空白，从而促进对各种糖缀合物以及构建它们的酶（糖基转移酶）和分解它们的酶（糖苷酶）的研究。在这里，我们概述了一个主要以化学为基础的研究项目，重点是创造使化学生物学工具。首先，使用标准显色或荧光底物对酶进行体外化学动力学研究，为糖苷水解酶如何作用于底物提供了至关重要的见解。然而，最终，这些酶在细胞的复杂环境中，在代谢物和蛋白质伴侣的存在下起作用。因此，我们将推进我们的开创性研究，合成明亮荧光共振能量转移（FRET）淬灭底物的一套糖苷酶。这些探针将可用于多种格式，以多种方式定量测量细胞内功能相关的人类酶的活性。其次，活细胞酶学是一个前沿领域，其目标是直接分析酶在活细胞的天然环境中的活性。然而，这一新兴领域一直受到一系列技术挑战的阻碍。在这里，我们的目标是开发两种糖苷酶的比例“笼”底物，并利用这些底物在活细胞中通过显微镜建立第一个详细的酶动力学研究。这项工作将为了解内源性细胞因子如何影响细胞内酶的功能奠定基础。第三，我们的目标是化学合成两种相关糖苷酶和糖基转移酶的“笼状”抑制剂。一旦这些最初无活性的笼状抑制剂被光照射，光保护基团就会丢失，有效的抑制剂就会释放出来。这些工具将允许对细胞和组织中的这些重要酶进行空间和时间控制。总之，这三个目标的进展将为观察和控制糖苷酶和糖基转移酶提供更好的工具。这些在细胞内使用的新工具将加速对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该适用于其他碳水化合物加工酶，实际上，更广泛地适用于其他超家族的酶。