Use of activity-based probes to study the action of glycosidases

开发新的方法来剖析碳水化合物在活细胞中的不同和奇妙的作用是现代细胞生物学、生物化学和医学面临的主要挑战。碳水化合物的巨大潜力反映在多糖的多种功能上；除了作为能源，碳水化合物在细胞和有机体结构、通讯信号和表观遗传修饰中发挥着核心作用。该项目将建立在称为糖苷酶的碳水化合物降解酶的基于活动的概况方面的最新进展的基础上；特别是在溶酶体储存疾病的背景下。这名学生将首先分析“下一代”探针与人类高谢病和法布里病酶的结合。三维结构将为改进的探针的合成提供信息。探测器将用于定量健康和疾病细胞中的酶活性，与Overkleeft和Aerts、Leiden合作，可能导致新诊断技术的开发。随着项目的发展，我们希望看到探针的概念推广到其他酶类和应用，最终，我们希望将这种技术与冷冻电子断层扫描方法相结合，以探测活细胞中的酶的活性。

The development of new approaches to dissect the diverse and wonderful roles for carbohydrates in living cells is a major challenge for modern cell biology, biochemistry and medicine. The colossal potential of carbohydrates is reflected in the multiplicity of functions for glycans; in addition to acting as energy sources, carbohydrates play central roles in cell and organism structure, communication signalling and epigenetic modification. This project will build upon recent developments in the activity-based profiling of carbohydrate-degrading enzymes termed gycosidases; notably in the context of lysosomal storage diseases. The student will initially analyse binding of "next-generation" probes to the human Gaucher and Fabry disease enzymes. 3-D structure will inform the synthesis of improved probes. Probes will be used to quantity enzyme activity in healthy and disease cells, working in collaboration with Overkleeft and Aerts, Leiden possibly leading to the development of new diagnostic technologies. As the project evolves, we expect to see the probe concept rolled-out to other enzyme classes and applications and ultimately, we would like to combine such techniques with cryo-electron tomography approaches to probe the activity of enzymes within living cells.