Doaa Samaha, Housam H. Hamdo, Xiaojing Cong, Fabian Schumacher, Sebastian Banhart, Öznur Aglar, Heiko Michael Möller, Dagmar Heuer, Burkhard Kleuser, Essa M. Saied, Christoph Arenz
- Ceramide transfer protein (CERT) mediates non-vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate-limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non-homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Forster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT-mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96-well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.
MetadatenAuthor details: | Doaa Samaha, Housam H. Hamdo, Xiaojing Cong, Fabian SchumacherORCiDGND, Sebastian BanhartORCiD, Öznur Aglar, Heiko Michael MöllerORCiDGND, Dagmar Heuer, Burkhard KleuserORCiDGND, Essa M. SaiedORCiD, Christoph Arenz |
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DOI: | https://doi.org/10.1002/chem.202003283 |
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ISSN: | 0947-6539 |
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ISSN: | 1521-3765 |
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Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/33047409 |
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Title of parent work (English): | Chemistry - a European journal |
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Publisher: | Wiley-VCH |
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Place of publishing: | Weinheim |
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Publication type: | Article |
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Language: | English |
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Date of first publication: | 2020/10/12 |
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Publication year: | 2020 |
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Release date: | 2023/02/02 |
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Tag: | Forster resonance energy transfer (FRET); enzyme assays; liposomes; sphingolipids; transport proteins |
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Volume: | 26 |
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Issue: | 70 |
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Number of pages: | 6 |
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First page: | 16616 |
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Last Page: | 16621 |
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Funding institution: | Deutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [AR; 376/10-1]; Government of Egypt; EXIST program of the BMBFFederal; Ministry of Education & Research (BMBF); School of Analytical Sciences; Adlershof (SALSA) - DFGGerman Research Foundation (DFG); Projekt DEAL |
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Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Ernährungswissenschaft |
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DDC classification: | 5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften |
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Peer review: | Referiert |
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Publishing method: | Open Access / Hybrid Open-Access |
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License (German): | CC-BY - Namensnennung 4.0 International |
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