Molekulare Charakterisierung der Interaktion zwischen Protein-Transmembrandomänen und Sphingolipiden.
Final Report Abstract
Sphingolipids are a major lipid class needed for the structural integrity of cellular membranes and as crucial regulators of many important cellular processes. Sphingolipids display a great variety of chemical structures, which are highly regulated, e.g. as a function of tissue distribution. As small molecules sphingolipids also need to interact with proteins to exert their effect. Here we examined the protein-lipid interaction of sphingolipids with different structures (species) with proteins in several ways. As lipids and sphingolipids in general interact with transmembrane proteins within the membrane, the total composition of these membranes is an important condition for their interaction. In two publications we looked at the lipid compositions of viruses. In order to be able to quickly analyze the data, we developed ShinyLipids, a web application to dynamically analyze lipidomes and their bulk lipid properties. To the end of detecting lipid-protein interactions on the species level we set out to detect protein transmembrane domain-lipid cross links that result from the interaction of proteins with photo-activatable lipids in vitro or in vitro. This is a challenging task because of the high hydrophobicity of the transmembrane domain on the one hand and the interacting lipid on the other hand. We established a MALDI method for detecting hydrophobic transmembrane domains, lipididated peptides and photo-crosslinks of transmembrane domains with a sphingolipid. Through our collaboration with Per Haberkant we participated in a publication describing pac-sphingosine as new chemical tool to find sphingolipid interacting proteins. A proteomic screen with pac-sphingosine in a mouse embryonic fibroblast cell line was used in which the sphingosine-1-phosphate lysase (S1PL) protein was knocked out, considerably enlarged the number of potential proteins interacting with sphingolipids. A S1PL knockout can be used to prevent the pac-labeling to leave the category of sphingolipids. To widen our toolbox we used the quickly rising CRSIPR technology to create a similar knock-out in the more accessible HeLa cell line and characterized it thoroughly as a cell line for protein-lipid interactions. Departing from the HeLa ΔSGPL1 background, we then created additional knock-outs of ceramide synthases, thereby creating HeLa cell lines habouring very different sphingolipid species that are now used in two different projects to examine the role of the fatty acid length in protein-sphingolipid interactions. We realized the great potential of the quickly emerging CRISPR technology to produce cells lines with different sphingolipid metabolism by knock-out of ceramide synthases and therefore turned our focused from the detection of protein-lipid intermediates with MS towards pac-sphingosine based screens in these newly generated cell lines.
Publications
- (2013). Comparative lipidomics analysis of HIV-1 particles and their producer cell membrane in different cell lines. Cellular Microbiology, 15(2), 292–304
Lorizate, M., Sachsenheimer, T., Glass, B., Habermann, A., Gerl, M. J., Kräusslich, H.-G., & Brugger, B.
(See online at https://doi.org/10.1111/cmi.12101) - (2014). Analysis of transmembrane domains and lipid modified peptides with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Anal Chem, 86(8), 3722–3726
Gerl, M. J., Sachsenheimer, T., Grzybek, M., Coskun, U., Wieland, F. T., & Brügger, B.
(See online at https://doi.org/10.1021/ac500446z) - Bifunctional Sphingosine for Cell-Based Analysis of Protein-Sphingolipid Interactions. ACS Chemical Biology, 2016, 11 (1), pp 222–230
Haberkant, P., Stein, F., Höglinger, D., Gerl, M. J., Brugger, B., Van Veldhoven, P. P., et al.
(See online at https://doi.org/10.1021/acschembio.5b00810)
