Chemical tools to investigate the role of sphingolipids in disease
[eng] Sphingolipids (SLs) encompass a large and diverse family of lipids that are essential constituents of cellular membranes. In addition to their structural functions, SLs are also emerging as important signalling molecules with an essential role in controlling different cellular processes such a...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/106625 |
| Acceso en línea: | https://hdl.handle.net/2445/106625 http://hdl.handle.net/10803/399916 |
| Access Level: | acceso abierto |
| Palabra clave: | Esfingolípids Membranes cel·lulars Enzims Sphingolipids Cell membranes Enzymes |
| Sumario: | [eng] Sphingolipids (SLs) encompass a large and diverse family of lipids that are essential constituents of cellular membranes. In addition to their structural functions, SLs are also emerging as important signalling molecules with an essential role in controlling different cellular processes such as cell proliferation, growth, migration, differentiation, senescence and apoptosis. Ceramide (Cer) and dihydroceramide (dhCer) are the central molecules of SLs metabolism. The bioactive role of dhCer has been recently described and it has been implicated in processes like autophagy. Alterations in SLs levels are strongly correlated with the initiation and progression of several diseases, such as type 2 diabetes, Alzheimer’s disease or different types of cancer. The metabolism of the SLs are catalyzed by specific enzymes, like dihydroceramide desaturase 1 (Des1), ceramidases (CDases), among others, and their study is important to understand the role of SLs. As mentioned above, experimental evidences suggest a role for dhCer in autophagy induction, but whether dhCer leads to prosurvival or lethal autophagy is subject of controversy. On the other hand, despite the advances in the development of Acid Ceramidase (AC) directed activity based probes (ABP), there is still a clear need for more specific and potent tools to characterize AC activity in cells. In this context, the objectives of this thesis are: 1. To validate the role of dihydroceramides as mediators of autophagy induction and outcome in cancer cell models. To achieve this objective, dhCer levels were induced to increase using two different types of compounds: Metabolic precursors and Des1 inhibitors. The first study was focused on the use of the metabolic intermediates of the de novo pathway, specifically 3-ketosphinganine (KSa) and its dideuterated analog at C4 (d2KSa) in three cancer cell lines. KSa and d2KSa are metabolized to produce high levels of dihydrosphingolipids in HGC27, T98G and U87MG cancer cells. In contrast, either direct C1 O-phosphorylation or N-acylation of d2KSa to produce dideuterated ketodihydrosphingolipids does not occur. Time-course experiments agree with sphinganine, sphinganine-1-phosphate and dihydroceramides being the mediators of autophagy stimulated by d2KSa. Enzyme inhibition studies support that inhibition of Des1 by 3-ketobases is caused by their dihydroceramide metabolites. However, this effect contributes to increasing dihydrosphingolipid levels only at short incubation times, since cells respond to long time exposure to 3-ketobases with Des1 overexpression. In the second study, the role of dhCer as inducers of autophagy was examined using drugs and pharmacological tools shown to decrease Des1 activity and reportedly able to stimulate autophagy. This study was carried out using glioblastoma T98G and U87MG cell lines and compounds like gamma- tocopherol (g-T), gamma-tocotrienol (g-TE), and the active site directed Des1 inhibitor XM462 were studied. g-TE and XM462 but not g-T induces autophagy concomitantly with dhCer build-up due to stimulation of Cer synthesis de novo and decreased Des1 activity. 2. To develop an activity based probe (ABP) for acid ceramidase (AC) detection in cells. In this study, we report on the activity of two SABRAC-based probes: a Bodipy-labeled analog (Bodipy-SOBRAC) and a clickable azido-substituted analog (N3C14SOBRAC) for further click derivatization with fluorescent alkynes. Both compounds were effective at detecting AC in cell lysates. |
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