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Synthetic Sphingolipid analogues : biophysical properties and subcellular localization

  • An interesting subclass of the SLs are Cers, the simplest SLs. Cers are assigned a special role within SLs because of their involvement in many cellular and biophysical processes.In literature Cers are describe to modulate many events in signaling including apoptosis. Besides its role as second messenger and therefore the involvement in many signal cascades, Cers are also known to be essential in physical modifications and structural alternations of membranes. Such regulatory functions on membrane formation are e.g. domain formation with other lipids (i.g. SM and Chol), phase separation with sterols (Chol), vesicular trafficking, fusion, membrane curvature fluidity and thickness and the induction of membrane leakiness. In contrast to phospholipids, Cers can move from one side of the membrane leaflet to the other, due to their strong hydrophobicity. This movement is called flip-flop or as transbilayer movement and is controversially discussed. Consequently, no exact value has been reported about the flip-flop property of Cers, which probably plays an important role during the transmission of an extra cellular signal through the membrane.In order to probe the biophysical properties of ceramides, a synthetic access to 1-thioceramides (1-SHCer) analogues with different N-acyl chain length has been developed in this study. With 1SHCer the flip-flop was investigated on pre-formed liposomes and the data indicated a very rapid flip-flop of Cers with a half time t1/2 <10s in raft- and non-raft like membrane models. Furthermore, the acyl chain length exhibited no measurable impact on the speed of the flip-flop. Utilizing the same probes the importance of hydrogen bond donor and acceptor properties of Cers upon interaction with sphingomyelin in the presence or absence of cholesterol (Chol) has been probed. Performed fluorescent quenching experiments (P.Slotte) proposed the following relative preference in interaction with pSM:pSM:DAGs > pSM:Cer > pSM:Chol > pSM: 1-pCerSH.Most strikingly, the importance of the 1-OH H-bond acceptor functionality to replace Chol around and above the melting temperature of pSM has been demonstrated. Recently, an unusual subclass of SLs, named 1-deoxysphingoids have come to the foreground, as biomarker for metabolic disorders. 1-doxSA is physiologically generated (10-40nM) due to substrate promiscuity of SPT and shown to be elevated in patients with metabolic disorders. In this study an organic synthetic access to fluorescent DSB derivatives was established, featuring a fluorescent moiety at the lipid tail, such as FITC 26. Comprehensive fluorescent studies of 26 revealed an unusual subcellular distribution. Exogenous 1-doxSA analogues, such as FB1 and 1-doxSA-FITC, enter via specific entry points. During the next few hours these lipids accumulate within the cytosol prior to N-acylation by CerS. Upon N-acylation, the newly formed 1-doxdhCer and its analogues insert into the ER membrane.The fluorescent probe and most likely FB1 analogues accumulate within the late endosomal and lysosomal system, probably via a direct connection with the ER. Analysis of the lipid metabolism of unlabeled 1-doxSA and FB1 revealed a strikingly similar behavior, pointing towards a common pharmacological effect. Complete consumption of TG within 24h in epithelia cells combined with GO analysis of 1-doxSA interacting lipids indicates significant modulation of fatty acid degradation, pointing towards regulation of the energy metabolism. This is in good agreement with the observed induction of autophagy. Together, this rapid and similar metabolic change of both 1-doxSA and FB1, points toward direct 1-doxSA head-group related lipid-protein interaction and less toward the influence of FB1 on CerS activity. This work suggests the biological significance of 1-doxSA as a primary nutrient sensor to maintain nutrient homeostasis and its role in the pathophysiology of metabolic diseases.
  • Ceramide stellen eine minor Komponente von biologischen Membranen dar, dennoch sind sie an einer Vielzahl von lebenswichtigen Zellfunktionen beteiligt. Die Bildung von Ceramiden, katalaysiert durch Sphingomyelinase, ist weiterhin involviert in der Modulation von Membranstruktur, im Vesikeltransport, an Membranusio/-teilung sowie an zellulären Signalvorgängen. Um die biophysikalischen Eigenschaften der Ceramide zu untersuchen, wurden synthetische 1-thioceramide Analoga entwickelt. Die 1-thioceramid Analoga stellen ein einzigartige Probe dar um die Bedeutung der Wasserstoffbrücken-Wechselwirkungen zwischen Ceramide und Sphingomyelin in Gegenwart oder bei Abwesenheit von Cholesterin zu untersuchen und zu bewerten. Desweiteren wurden innerhalb dieser Arbeit Synthesestrategien für Fuminosin- und 1-deoxysphingosin analogas entwickelt. Diese fluoreszierenden Verbindungen kamen in Zellstudien zum Einsatz und den noch weitestgehend unbekannten subzellulären Transport der 1-deoxysphingolipide zu untersuchen. Hierfür wurden Zellen mit der fluoreszierenden Verbindung markiert und deren Transport innerhalb der Zelle mittels konfokale -Laser-Scanning-Mikroskopie beobachtet. Um mögliche Bindungspartner von 1-Deoxysphinganie aufzudecken, sowie die weitere Charakterisierung des Signalweges, wurden Metabolismusstudien an verschiedenen Zelllinien durchgeführt.

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Author: Sabrina Kargoll
Title Additional (German):Synthetische Sphingolipide : biophysikalische Eigenschaften und subzelluläre Lokalisation
Advisor:Prof. Dr. Winfried Hinrichs
Document Type:Doctoral Thesis
Date of Publication (online):2016/07/04
Granting Institution:Ernst-Moritz-Arndt-Universität, Mathematisch-Naturwissenschaftliche Fakultät (bis 31.05.2018)
Date of final exam:2016/04/13
Release Date:2016/07/04
Tag:1-deoxy sphingolipids, transbilayer lipid (flip-flop) motion
GND Keyword:Ceramide, Diabetes mellitus, Sphingolipide
Faculties:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie und Biochemie
DDC class:500 Naturwissenschaften und Mathematik / 540 Chemie