Universität zu Köln

Gerl, Mathias J., Bittl, Verena, Kirchner, Susanne, Sachsenheimer, Timo, Brunner, Hanna L., Luechtenborg, Christian, Oezbalci, Cagakan, Wiedemann, Hannah, Wegehingel, Sabine, Nickel, Walter, Haberkant, Per, Schultz, Carsten ORCID: 0000-0002-5824-2171, Krueger, Marcus ORCID: 0000-0003-2008-4582 and Bruegger, Britta (2016). Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions. PLoS One, 11 (4). SAN FRANCISCO: PUBLIC LIBRARY SCIENCE. ISSN 1932-6203

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Abstract

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Gerl, Mathias J. UNSPECIFIED UNSPECIFIED UNSPECIFIED Bittl, Verena UNSPECIFIED UNSPECIFIED UNSPECIFIED Kirchner, Susanne UNSPECIFIED UNSPECIFIED UNSPECIFIED Sachsenheimer, Timo UNSPECIFIED UNSPECIFIED UNSPECIFIED Brunner, Hanna L. UNSPECIFIED UNSPECIFIED UNSPECIFIED Luechtenborg, Christian UNSPECIFIED UNSPECIFIED UNSPECIFIED Oezbalci, Cagakan UNSPECIFIED UNSPECIFIED UNSPECIFIED Wiedemann, Hannah UNSPECIFIED UNSPECIFIED UNSPECIFIED Wegehingel, Sabine UNSPECIFIED UNSPECIFIED UNSPECIFIED Nickel, Walter UNSPECIFIED UNSPECIFIED UNSPECIFIED Haberkant, Per UNSPECIFIED UNSPECIFIED UNSPECIFIED Schultz, Carsten UNSPECIFIED orcid.org/0000-0002-5824-2171 UNSPECIFIED Krueger, Marcus UNSPECIFIED orcid.org/0000-0003-2008-4582 UNSPECIFIED Bruegger, Britta UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-278393
DOI:	10.1371/journal.pone.0153009
Journal or Publication Title:	PLoS One
Volume:	11
Number:	4
Date:	2016
Publisher:	PUBLIC LIBRARY SCIENCE
Place of Publication:	SAN FRANCISCO
ISSN:	1932-6203
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language SPHINGOSINE 1-PHOSPHATE; CLICK CHEMISTRY; SPHINGOLIPID INTERACTIONS; QUANTITATIVE-ANALYSIS; MEMBRANE; CRISPR-CAS9; METABOLISM; LIVER; DEHYDROGENASE; PURIFICATION Multiple languages Multidisciplinary Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/27839