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Title: Chemical tools to investigate the role of sphingolipids in disease
Author: Ordóñez Vivanco, Yadira
Director: Fabriàs Domingo, Gemma
Triola i Guillem, Gemma
Grandas Segarra, Anna
Keywords: Esfingolípids
Membranes cel·lulars
Cell membranes
Issue Date: 16-Sep-2016
Publisher: Universitat de Barcelona
Abstract: [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.
[spa] Evidencias experimentales sugieren un papel de las dihidroceramidas (dhCer) en la inducción de la autofagia, pero si las dhCer conducen a autofagia protectora o letal es objeto de controversia. Por otro lado, a pesar de los avances en el desarrollo de sondas basadas en actividad (ABP) para el marcaje de la ceramidasa ácida (AC), todavía hay una clara necesidad de desarrollar herramientas más específicas y potentes para caracterizar la actividad de la AC en las células. En este contexto, los objetivos de este trabajo de tesis son: 1. Validar el papel de las dhCer como mediadores de la inducción de autofagia en modelos celulares de cáncer. Para llevar a cabo éste objetivo, se ha procedido a aumentar los niveles de dhCer mediante el uso de dos tipos diferentes de herramientas farmacológicas: precursores metabólicos y inhibidores de la Dihidroceramida desaturasa (Des1). En la primera aproximación, se utilizó cetoesfinganina (KSa) y su análogo dideuterado en C4 (d2KSa) como precursores metabólicos de dhCer. Tanto la KSa como la d2KSa se metabolizaron para producir altos niveles de dihidroesfingolípidos (dhSLs) en las líneas celulares tumorales HGC27, T98G y U87MG. Experimentos realizados a diferentes tiempos de tratamiento sugieren que la esfinganina, la esfinganina-1-fosfato y la dihidroceramida son probablemente los mediadores de la autofagia producida tras el tratamiento con d2KSa. La inhibición de la Des1 observada con estas 3-cetobases es causada por su metabolización a dihidroceramida. En la otra aproximación dirigida a estudiar el papel de las dhCer en la activación de la autofagia se utilizaron compuestos inhibidores de la Des1 cómo gamma-tocotrienol (g-TE) y XM462, capaces de incrementar los niveles intracelulares de dhCer, en las líneas de glioblastoma U87MG y T98G. Todos los compuestos estudiados excepto el gamma-tocoferol (g-T) indujeron el flujo autofágico de forma concomitante con la acumulación de dhCer, la cual ocurrió por estimulación de la biosíntesis de novo y disminución de la actividad Des1. 2. Desarrollar una sonda basada en la actividad enzimática (ABP) para la detección de ceramidasa ácida (AC). En éste estudio, se ha analizado la actividad de diferentes análogos del SABRAC. Estos compuestos son inhibidores potentes de la AC y además presentan diferentes grupos funcionales o fluorescentes que permiten su uso como ABP para el análisis de enzima activa. Los dos compuestos han sido efectivos para la detección de AC en lisados celulares.
Appears in Collections:Tesis Doctorals - Departament - Química Inorgànica i Orgànica

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