组会讲课人员:胥宁格
Quantifying lysosomal glycosidase activity within cells using bis-acetal substrates
使用双缩醛底物量化细胞内的溶酶体糖苷酶活性
主讲人:胥宁格
Nature Chemical Biology | VOL 18 | March 2022 | 332–341 https://doi.org/10.1038/s41589-021-00960-x
Abstract:Understanding the function and regulation of enzymes within their physiologically relevant milieu requires quality tools that report on their cellular activities. Here we describe a strategy for glycoside hydrolases that overcomes several limitations in the field, enabling quantitative monitoring of their activities within live cells. We detail the design and synthesis of bright and modularly assembled bis-acetal-based (BAB) fluorescence-quenched substrates, illustrating this strategy for sensitive quantitation of disease-relevant human α-galactosidase and α-N-acetylgalactosaminidase activities. We show that these substrates can be used within live patient cells to precisely measure the engagement of target enzymes by inhibitors and the efficiency of pharmacological chaperones, and highlight the importance of quantifying activity within cells using chemical perturbogens of cellular trafficking and lysosomal homeostasis. These BAB substrates should prove widely useful for interrogating the regulation of glycosidases within cells as well as in facilitating the development of therapeutics and diagnostics for this important class of enzymes.
摘要:了解酶在其生理相关环境中的功能和调节需要报告其细胞活动的质量工具。在这里,作者描述了一种糖苷水解酶的策略,该策略克服了该领域的一些限制,从而能够定量监测它们在活细胞内的活动。作者详细介绍了明亮且模块化组装的双缩醛基 (BAB) 荧光猝灭底物的设计和合成,阐明了这种灵敏定量与疾病相关的人类α-半乳糖苷酶和α-N-乙酰半乳糖苷酶活性的策略。作者证明,这些底物可以在活的患者细胞内用于精确测量抑制剂与靶酶的结合以及药理伴侣的效率,并强调使用细胞运输和溶酶体内稳态的化学扰动因子量化细胞内活性的重要性。这些BAB底物对于研究细胞内糖苷酶的调节以及促进这类重要酶的治疗学和诊断学的发展具有广泛的用途。