A fluorogenic substrate for the detection of lipid amidases in intact cells
Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2024-03-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227524000257 |
| _version_ | 1797238139953086464 |
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| author | Mireia Casasampere Johnson Ung Alejandro Iñáñez Carine Dufau Kazuhito Tsuboi Josefina Casas Su-Fern Tan David J. Feith Nathalie Andrieu-Abadie Bruno Segui Thomas P. Loughran, Jr. José Luis Abad Gemma Fabrias |
| author_facet | Mireia Casasampere Johnson Ung Alejandro Iñáñez Carine Dufau Kazuhito Tsuboi Josefina Casas Su-Fern Tan David J. Feith Nathalie Andrieu-Abadie Bruno Segui Thomas P. Loughran, Jr. José Luis Abad Gemma Fabrias |
| author_sort | Mireia Casasampere |
| collection | DOAJ |
| description | Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 μM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 μM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 μM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells. |
| first_indexed | 2024-04-24T17:30:54Z |
| format | Article |
| id | doaj.art-cbc54b7eb8e74f508add3ca8fe1b0079 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-04-24T17:30:54Z |
| publishDate | 2024-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-cbc54b7eb8e74f508add3ca8fe1b00792024-03-28T06:36:41ZengElsevierJournal of Lipid Research0022-22752024-03-01653100520A fluorogenic substrate for the detection of lipid amidases in intact cellsMireia Casasampere0Johnson Ung1Alejandro Iñáñez2Carine Dufau3Kazuhito Tsuboi4Josefina Casas5Su-Fern Tan6David J. Feith7Nathalie Andrieu-Abadie8Bruno Segui9Thomas P. Loughran, Jr.10José Luis Abad11Gemma Fabrias12Department of Biological Chemistry, Research Unit on BioActive Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, SpainDivision of Hematology and Oncology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA, USA; Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA, USADepartment of Biological Chemistry, Research Unit on BioActive Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, SpainINSERM UMR 1037, Cancer Research Center of Toulouse (CRCT), Toulouse, France; Equipe Labellisée Fondation ARC pour la recherche sur le cancer, Toulouse, FranceDepartment of Pharmacology, Kawasaki Medical School, Kurashiki, Okayama, JapanDepartment of Biological Chemistry, Research Unit on BioActive Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain; CIBEREHD, Madrid, SpainDivision of Hematology and Oncology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA, USA; University of Virginia Cancer Center, University of Virginia School of Medicine, Charlottesville, VA, USADivision of Hematology and Oncology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA, USA; University of Virginia Cancer Center, University of Virginia School of Medicine, Charlottesville, VA, USAINSERM UMR 1037, Cancer Research Center of Toulouse (CRCT), Toulouse, France; Equipe Labellisée Fondation ARC pour la recherche sur le cancer, Toulouse, FranceINSERM UMR 1037, Cancer Research Center of Toulouse (CRCT), Toulouse, France; Equipe Labellisée Fondation ARC pour la recherche sur le cancer, Toulouse, France; Université Toulouse III - Paul Sabatier, Toulouse, FranceDivision of Hematology and Oncology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA, USA; University of Virginia Cancer Center, University of Virginia School of Medicine, Charlottesville, VA, USADepartment of Biological Chemistry, Research Unit on BioActive Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain; For correspondence: José Luis Abad; Gemma FabriasDepartment of Biological Chemistry, Research Unit on BioActive Molecules, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain; CIBEREHD, Madrid, Spain; Spanish National Research Council (CSIC)’s Cancer Hub, Madrid, Spain; For correspondence: José Luis Abad; Gemma FabriasLipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 μM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 μM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 μM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells.http://www.sciencedirect.com/science/article/pii/S0022227524000257sphingolipidsceramideslipidsenzymologychemical synthesisN-palmitoylethanolamine |
| spellingShingle | Mireia Casasampere Johnson Ung Alejandro Iñáñez Carine Dufau Kazuhito Tsuboi Josefina Casas Su-Fern Tan David J. Feith Nathalie Andrieu-Abadie Bruno Segui Thomas P. Loughran, Jr. José Luis Abad Gemma Fabrias A fluorogenic substrate for the detection of lipid amidases in intact cells Journal of Lipid Research sphingolipids ceramides lipids enzymology chemical synthesis N-palmitoylethanolamine |
| title | A fluorogenic substrate for the detection of lipid amidases in intact cells |
| title_full | A fluorogenic substrate for the detection of lipid amidases in intact cells |
| title_fullStr | A fluorogenic substrate for the detection of lipid amidases in intact cells |
| title_full_unstemmed | A fluorogenic substrate for the detection of lipid amidases in intact cells |
| title_short | A fluorogenic substrate for the detection of lipid amidases in intact cells |
| title_sort | fluorogenic substrate for the detection of lipid amidases in intact cells |
| topic | sphingolipids ceramides lipids enzymology chemical synthesis N-palmitoylethanolamine |
| url | http://www.sciencedirect.com/science/article/pii/S0022227524000257 |
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