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Chang, Young Tae
복잡계 자기조립 연구단
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Click and count: specific detection of acid ceramidase activity in live cells

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Title
Click and count: specific detection of acid ceramidase activity in live cells
Author(s)
Casasampere, Mireia; Izquierdo, Eduardo; Casas, Josefina; Abad, Jose Luis; Xiao Liu; Xu, Ruijuan; Mao, Cungui; Young-Tae Chang; Delgado, Antonio; Fabrias, Gemma
Publication Date
2020-12
Journal
Chemical Science, v.11, no.48, pp.13044 - 13051
Publisher
Royal Society of Chemistry
Abstract
The use of intact cells in medical research offers a number of advantages over employing cell-free systems. In diagnostics, cells isolated from liquid biopsies can be directly used, speeding up the time of analysis and diminishing the risk of protein degradation by sample manipulation. In drug discovery, studies in live cells take into account aspects neglected in cell-free systems, such as uptake, metabolization, and subcellular concentration by compartmentalization of potential drug candidates. Therefore, probes for studies in cellulo are of paramount importance. Acid ceramidase (AC) is a lysosomal enzyme that hydrolyses ceramides into sphingoid bases and fatty acids. The essential role of this enzyme in the outburst and progress of several diseases, some of them still incurable, is well sustained. Despite the great clinical relevance of AC as a biomarker and therapeutic target, the specific monitoring of AC activity in live cells has remained elusive due to the concomitant existence of neutral and alkaline ceramidases. In this work, we report that 1-deoxydihydroceramides are exclusively hydrolysed by AC. Using N-octanoyl-18-azidodeoxysphinganine as a probe and a BODIPY-substituted bicyclononyne, we show the click-reliant predominant staining of lysosomes, with extra-lysosomal labeling also occurring in some cells. Importantly, using pharmacological and genetic tools together with high resolution mass spectrometry, we demonstrate that both lysosomal and extra-lysosomal staining are AC-dependent. These findings are translated into the specific flow cytometry monitoring of AC activity in intact cells, which fills an important gap in the field of diseases linked to altered AC activity.
URI
https://pr.ibs.re.kr/handle/8788114/9099
DOI
10.1039/d0sc03166f
ISSN
2041-6520
Appears in Collections:
Center for Self-assembly and Complexity(복잡계 자기조립 연구단) > 1. Journal Papers (저널논문)
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