Evaluierung der Lipidmuster S-acylierter Proteine und Azido-Ceramid Derivate zur proteomweiten Identifizierung neuer C16- Ceramid bindender Proteine
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2017
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Part 01: A Bioorthogonal Probe for Quantitative Profiling of Protein S-Acylation We have developed a chemical probe that enables the accurate and systematic identification of Sbound fatty acids. The analysis of the S-acylome in different cell lines as well as in an enriched N-Ras revealed that proteins are modified with fatty acids of various chain lengths and structures. These previously overlooked fatty acid modifications may have a key role in regulating protein localization and function. Hence, atomic force microscopy studies and FRET-based kinetics assays indicated that the unsaturated N-Ras presents an increased tendency toward clustering and higher insertion kinetic rate constants compared to that of its saturated counterpart. The technique could be also applied to investigate the role of CLN3 as a potential fatty acid desaturase. Indeed, overexpression of CLN3 results in all the cases in a significant increase in C16:1 levels. This effect was most marked for N-Ras compared to the whole S-acylome, which it may be explained by the substrate specificity of CLN3 or its particular subcellular localization. We are convinced that the developed methodology may strongly contribute to a better understanding of S-acylated proteins. Part 02: Azido-tagged Sphingolipids for the proteome-wide identification of novel C16-ceramidebinding proteins To generate a candidate list of ceramide-interacting proteins we applied two methods, a mass spectrometry-based proteomic approach and a proteome microarray. 214 and 127 protein candidates were identified respectively, and 24 were found in both data sets. We initially chose potential candidates for testing on the basis of its potential implication in ceramide-mediated pathways. Binding could be confirmed for: AIFm2, APP, BUD31, Gal-1 and Gal-3, mTOR and PPT1. The specificity of those proteins for ceramide species with different chain lengths has been also analysed using a pull-down and a competition assay, observing a dose-dependent decrease in the interaction of the lipid with the studied proteins. To further validate the binding of ceramide to the identified proteins we employed a cellular thermal shift assay. Treatment of cell lysates with C16-Cer resulted in a slighter stabilization of APP, BUD31 and PPT1 and an appreciable increase of melting point temperatures for the other investigated proteins. Finally, the direct interaction of C16-Cer was investigated in vitro using a DSF and recombinantly expressed proteins. AIFm2, BcL-xL, Gal-1 were selected and direct interaction with variable affinities could be proved in all the cases, whereas no binding could be observed when AIFm2 and Gal-1 were titrated with increasing concentrations of palmitic acid, or when C-16-Cer was tested against Son of Sevenless (SOS) protein as a negative control. Future experiments will be required to elucidate how ceramide binding affects protein activity, if it relies on the modulation of protein localization such as the targeting of LC3B to mitochondria, or if, in turn, it directly regulates protein function such as the activation of the protein phosphatase PP2A.
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Lipidmuster, Ceramide