المؤلفون: 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

المصدر: Journal of Lipid Research, Vol 65, Iss 3, Pp 100520- (2024)

مصطلحات موضوعية: sphingolipids, ceramides, lipids, enzymology, chemical synthesis, N-palmitoylethanolamine, Biochemistry, QD415-436

الوصف: 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.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S0022227524000257Test; https://doaj.org/toc/0022-2275Test

الوصول الحر: https://doaj.org/article/cbc54b7eb8e74f508add3ca8fe1b0079Test

المؤلفون: Mireia Casasampere, Núria Bielsa, Daniel Riba, Laura Bassas, Ruijuan Xu, Cungui Mao, Gemma Fabriàs, José-Luis Abad, Antonio Delgado, Josefina Casas

المصدر: Journal of Lipid Research, Vol 60, Iss 6, Pp 1174-1181 (2019)

مصطلحات موضوعية: substrate, sphingolipids, umbelliferone, ceramides, Biochemistry, QD415-436

الوصف: New fluorogenic ceramidase substrates derived from the N-acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl chain can be used as substrates to determine alkaline ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S0022227520322574Test; https://doaj.org/toc/0022-2275Test

الوصول الحر: https://doaj.org/article/048c0511ba6143f3ae222eeae22fc56cTest

المؤلفون: Francesca Cingolani, Fabio Simbari, Jose Luis Abad, Mireia Casasampere, Gemma Fabrias, Anthony H. Futerman, Josefina Casas

المصدر: Journal of Lipid Research, Vol 58, Iss 8, Pp 1500-1513 (2017)

مصطلحات موضوعية: sphingolipid, liquid chromatography-mass spectrometry, methuosis, autophagy, anticancer drug, Biochemistry, QD415-436

الوصف: Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S0022227520337780Test; https://doaj.org/toc/0022-2275Test

الوصول الحر: https://doaj.org/article/941fee4dc31a45058d91b6e7b3dc22ceTest

المؤلفون: Mireia Casasampere, Luz Camacho, Francesca Cingolani, Josefina Casas, Meritxell Egido-Gabás, José Luís Abad, Carmen Bedia, Ruijuan Xu, Kai Wang, Daniel Canals, Yusuf A. Hannun, Cungui Mao, Gemma Fabrias

المصدر: Journal of Lipid Research, Vol 56, Iss 10, Pp 2019-2028 (2015)

مصطلحات موضوعية: ceramides, lipids, mass spectrometry, sphingolipids, enzymology, umbelliferone, Biochemistry, QD415-436

الوصف: Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N-acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N-acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N-acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S0022227520309779Test; https://doaj.org/toc/0022-2275Test

الوصول الحر: https://doaj.org/article/04c50654b16444fbb2d9d2d519fb9fb9Test

المؤلفون: Francesca Cingolani, Mireia Casasampere, Pol Sanllehí, Josefina Casas, Jordi Bujons, Gemma Fabrias

المصدر: Journal of Lipid Research, Vol 55, Iss 8, Pp 1711-1720 (2014)

مصطلحات موضوعية: autophagy, cell cycle, enzyme inhibition, molecular docking, mass spectrometry, reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase, Biochemistry, QD415-436

الوصف: Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S0022227520353347Test; https://doaj.org/toc/0022-2275Test

الوصول الحر: https://doaj.org/article/1ad82e157e494d40905c707db937cc90Test

المؤلفون: Fabio Simbari, José Luis Abad, Anthony H. Futerman, Francesca Cingolani, Gemma Fabriàs, Mireia Casasampere, Josefina Casas

المساهمون: Ministerio de Ciencia e Innovación (España), Fabriàs, Gemma [0000-0001-7162-3772], Fabriàs, Gemma

المصدر: Journal of Lipid Research, Vol 58, Iss 8, Pp 1500-1513 (2017)
Digital.CSIC. Repositorio Institucional del CSIC
instname

مصطلحات موضوعية: 0301 basic medicine, Programmed cell death, Ceramide, autophagy, Cell Survival, Acylation, Apoptosis, Vacuole, QD415-436, Anticancer drugs, Biochemistry, Sphingolipid, Wortmannin, 03 medical and health sciences, chemistry.chemical_compound, Endocrinology, methuosis, Sphingosine, Stomach Neoplasms, Cell Line, Tumor, Autophagy, Humans, anticancer drug, Liquid chromatography-mass spectrometry, Ceramide synthase, Research Articles, liquid chromatography-mass spectrometry, Methuosis, Cell Death, Cell Biology, Cell biology, 030104 developmental biology, chemistry, Vacuoles, Cancer cell, Pinocytosis, sphingolipid, Oxidoreductases

الوصف: Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.
This work was supported by the Ministerio de Ciencia e Innovaci?n (Project CTQ2014-54743-R) and a predoctoral contract from Generalitat de Catalunya to F.C. A.H.F. is the Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute of Science. The authors thank Prof. R. Ghidoni and Dr. V. Gagliostro (University of Milan) for preliminary LC3-II data and Prof. A. Delgado for helpful discussions. The authors also thank E. Dalmau for excellent technical assistance.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::247310c93ee16332816f4ac5001e2086Test
http://www.sciencedirect.com/science/article/pii/S0022227520337780Test

المؤلفون: Francesca Cingolani, Carmen Bedia, Ruijuan Xu, Mireia Casasampere, Yusuf A. Hannun, José Luis Abad, Meritxell Egido-Gabás, Kai Wang, Cungui Mao, Josefina Casas, Daniel Canals, Luz Camacho, Gemma Fabriàs

المساهمون: Ministerio de Economía y Competitividad (España), Fabriàs, Gemma [0000-0001-7162-3772], Bedia, Carmen [0000-0003-4584-5843], Fabriàs, Gemma, Bedia, Carmen

المصدر: Digital.CSIC. Repositorio Institucional del CSIC
instname
Journal of Lipid Research, Vol 56, Iss 10, Pp 2019-2028 (2015)

مصطلحات موضوعية: Ceramide, Stereochemistry, Acylation, enzymology, Ceramidases, QD415-436, Alkaline Ceramidase, Ceramides, Biochemistry, Amidase, Substrate Specificity, lipids, chemistry.chemical_compound, Mice, Structure-Activity Relationship, umbelliferone, Endocrinology, Coumarins, Neutral Ceramidase, Methods, Animals, Humans, Fluorescent Dyes, mass spectrometry, chemistry.chemical_classification, Sphingolipids, ceramides, sphingolipids, Hydrolysis, Cell Biology, HCT116 Cells, Acid ciramidase, Enzyme, HEK293 Cells, chemistry, Ceramidase activity, Gene Knockdown Techniques, Microsome, Erratum, HeLa Cells

الوصف: Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N -acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N -acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N -acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.
This work was supported, in whole or in part, by the Spanish Ministry of Economy and Competitiveness (Grants SAF2011-22444 and CTQ2014-54743-R to G.F.), the Fundació la Marató de TV3 (Grants 112130 to J.C. and 112132), Agència de Gestió d’Ajuts Universitaris i de Recerca of Generalitat de Catalunya (Grant 2009SGR1072 to G.F.), National Cancer Institute (Grant NCI PO1 97132 to Y.A.H.), and National Institutes of Health (Grant R01CA163825 to C.M.). L.C. and F.C. were supported by predoctoral fellowships and C.B. was supported by postdoctoral contracts awarded by the Generalitat de Catalunya.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::7f588f5c644420e1808fb7870de940b9Test
https://europepmc.org/articles/PMC4583077Test/