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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717678
• 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。