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Fluorescence-Quenched Substrates for Live Cell Imaging of Human Glucocerebrosidase Activity

Resource type
Date created
2015-01-05
Authors/Contributors
Author: He, Xu
Abstract
Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glucosyl ceramide in lysosomes and the development of the lysosomal storage disease known as Gaucher’s disease. More recently, mutations in the GBA1 gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinson’s disease (PD). Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of live cells. Indeed, visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, has proven problematic within live mammalian cells. Here we describe the successful modular design and synthesis of fluorescence-quenched substrates for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (∼99.9%) fluorescent substrates also permit assessment of GCase inhibition in live cells by either confocal microscopy or high content imaging. Such substrates should enable improved understanding of GCase in situ as well the optimization of small-molecule chaperones for this enzyme. These findings also suggest routes to generate fluorescence-quenched substrates for other mammalian glycoside hydrolases for use in live cell imaging.
Document
Identifier
DOI: 10.1021/ja5106738
Published as
Yadav, A. K., Shen, D. L., Shan, X., He, X., Kermode, A. R., & Vocadlo, D. J. (2015). Fluorescence-Quenched Substrates for Live Cell Imaging of Human Glucocerebrosidase Activity. Journal of the American Chemical Society, 137(3), 1181–1189. https://doi.org/10.1021/ja5106738
Publication title
Journal of the American Chemical Society
Document title
Fluorescence-Quenched Substrates for Live Cell Imaging of Human Glucocerebrosidase Activity
Date
2015
Volume
137
Issue
3
First page
1181
Last page
1189
Copyright statement
Copyright is held by the author(s).
Scholarly level
Peer reviewed?
Yes
Language
English

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