New Fluorogenic Probes for Neutral and Alkaline Ceramidases

    March 2019 in “ Journal of lipid research ”
    Mireia Casasampere, Núria Bielsa, Daniel Riba, Laura Bassas, Ruijuan Xu, Cungui Mao, Gemma Fabriàs, José Luı́s Abad, Antonio Delgado, Josefina Casas
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    Studysummary This study reports new fluorogenic ceramidase substrates and highlights RBM14C24:1 as an efficient substrate for neutral ceramidase, while RBM15C18:1 is the best probe for measuring ACER1 and ACER2 activities, potentially aiding high-throughput screening for ceramidase inhibitors. Our plain-language summary of this paper — not a Tressless recommendation.
    The document from June 2019 details the creation of new fluorogenic probes, RBM14 and RBM15, designed to measure the activity of neutral and alkaline ceramidases, which play a role in sphingolipid metabolism. The study found that the RBM14C24:1 probe was a highly selective substrate for human neutral ceramidase, while the RBM15C18:1 probe was the most effective for ACER1 and ACER2 ceramidases. These probes are important for developing high-throughput screening methods for ceramidase inhibitors with potential therapeutic applications. The study also provided kinetic parameters for the hydrolysis of substrates by neutral ceramidase, with KM values ranging from 8 to over 250 µM and Kcat/KM values from 15.4 to 546.5 M⁻¹s⁻¹, indicating efficient enzyme-substrate interactions. The effectiveness of the probes was confirmed through multiple experiments with triplicates, showing significant differences in umbelliferone release under various conditions and between different ceramidases (P < 0.05). The research was supported by the Spanish Ministry of Science, Innovation and Universities, with contributions from Alex García and Pedro Rayo.
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