Chemical approaches to the study of the ceramide synthase activity
[eng] Sphingolipids (SLs) are one of the major classes of lipids in eukaryotes. In addition to being essential structural components of cell membranes, SLs also play capital roles as signalling molecules. Ceramides (Cer) are a family of bioactive SLs consisting of a long chain base (LCB), known as t...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2020 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/176392 |
| Acceso en línea: | https://hdl.handle.net/2445/176392 http://hdl.handle.net/10803/671426 |
| Access Level: | acceso abierto |
| Palabra clave: | Esfingolípids Lípids Proteòmica Sphingolipids Lipids Proteomics |
| Sumario: | [eng] Sphingolipids (SLs) are one of the major classes of lipids in eukaryotes. In addition to being essential structural components of cell membranes, SLs also play capital roles as signalling molecules. Ceramides (Cer) are a family of bioactive SLs consisting of a long chain base (LCB), known as the sphingoid base, linked to a fatty acid (FA) of variable chain length via an amide bond. Due to their metabolic inter-relations with other SL species, Cer are considered key intermediates in the SL pathway. Cer are important second messengers in several cellular processes including apoptosis, autophagy, cell differentiation and sensescence. Ceramide synthases (CerS) are a group of enzymes, primarily localised at the endoplasmic reticulum, that catalyse the N–acylation of sphingoid bases such as sphingosine (So) and dihydrosphingosine (dhSo), using acyl CoA thioesters of variable chain lengths, to afford Cer and dihydroceramides (dhCer), respectively. Six isoforms of CerS (CerS1–6) have been identified in mammals. Each CerS isoform utilizes a small subset of FA-CoAs of defined chain lengths and, thus, each of them produces specific Cer populations. In the recent years, it has become apparent that Cer with different acyl chains vary in their biophysical properties and in the signalling pathways they participate. Furthermore, Cer with defined acyl chain lengths have been found to be implicated in the onset of a variety of human diseases, including cancer, type-2 diabetes mellitus, Alzheimer’s disease, multiple sclerosis and cardiomyopathy. In this context, the development of appropriate tools to study the activity of CerS enzymes, which is crucial to decipher the molecular mechanisms by which Cer elicit their effects, was the ultimate goal of the present doctoral thesis. The first part of this thesis was devoted to the development of a new CerS activity assay based on the Förster Resonance Energy Transfer (FRET) phenomenon. To that end, we designed and synthesized a series of fluorescently labelled (or labelable) 1-deoxy sphingoid probes derived from spisulosine, a small library of clickable FA analogues of different chain lengths, and a collection of bicyclo[6.1.0]nonyne (BCN) or 1,2,4,5-tetrazine (Tz) based fluorescent reagents. The absorption and fluorescence emission properties of these compounds was thoroughly studied in various solvents by means of cuvette-based experiments. Based on these studies, we anticipated that a highly efficient FRET process would take place between the donor-acceptor fluorophore pairs that had been selected, namely MCC/NBD and NBD/NR. Next, the metabolic incorporation of the different spisulosin-based probes and the FA analogues was evaluated in various biological contexts. Mass spectrometry analysis evidenced an extensive metabolization of the synthetic LCB probes and the FA analogues by CerS enzymes to form the corresponding Cer metabolites. Unfortunately, the FA analogues were also incorporated into other lipidic metabolic pathways, resulting in the generation of a strong fluorescence background after the fluorescent labelling reactions. Our different attempts to solve this issue were unfruitful and, thus, the development of the FRET based fluorescence assay to determine the CerS activity could not be achieved. The second part of this thesis was aimed at the development of new click-formed proteolysis targeting chimeras (CLIPTACs) targeting the ubiquitination and proteasomal degradation of CerS, as an alternative to small molecule inhibitors for the modulation of the CerS activity. To this end, we designed and synthesized four BCN derivatives containing known ligands for recruiting different E3 ubiquitin ligases. These BCN-tagged E3 ligase recruiters will be used in future studies in combination with an azido-functionalized analogue of the CerS substrate Jaspine B to obtain the desired CLIPTACs. |
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