Eicosanoid biosynthesis: getting the full picture
Lipidomics Gateway (28 March 2012) [doi:10.1038/lipidmaps.2012.9]
Quantification at the protein level of the changes in eicosanoid biosynthetic enzymes induced by Toll-like receptor-4 signalling adds to the existing metabolomic and genomic analyses to complete the full overview of the eicosanoid pathway.
A fully-integrated genomic, proteomic and metabolomic analysis of the eicosanoid biochemical pathway in RAW 264.7 macrophages is now complete following the profiling of the abundance of proteins involved in the biosynthesis of these bioactive lipid mediators carried out by Sabidó et al.
Eicosanoids, which have important functions in cell signalling and inflammation, are derived from arachidonic acid through a number of biosynthetic pathways. In the present study, the results of which complement transcriptomic and metabolite fluxomic data obtained previously by the same group, multiple reaction monitoring (MRM) was used to analyze changes in the levels of eicosanoid biosynthetic proteins. The MRM approach enables specific peptides of interest to be detected from within a sample by mass spectrometry, thereby facilitating their quantification. Sabidó et al. began by developing MRM assays for 79 proteins involved in eicosanoid biosynthesis; assays for a further 2 proteins could not be developed. In itself, this spectral library provides a valuable tool for the rapid quantification of multiple eicosanoid-related proteins in response to different conditions.
The researchers used these assays to study the effect of Kdo2-lipid A (KLA), an agonist of Toll-like receptor 4 (TLR-4), on the eicosanoid-related proteome. They were able to detect and quantify 29 proteins involved in eicosanoid biosynthesis in extracts prepared from macrophages at regular time points over a 24-hour period following KLA treatment; 18 of these proteins showed significant changes in abundance.
Confirming the importance of cyclooxygenase-2 (COX-2) in the macrophage inflammatory response to KLA, levels of this enzyme, which catalyzes the rate-limiting step in prostaglandin biosynthesis, increased over 32-fold compared to basal levels 8–24 hours following treatment, reflecting previously reported changes in COX-2 transcript levels. Furthermore, the abundance of several enzymes involved in prostaglandin breakdown was decreased. By contrast, protein levels of leukotriene A4 hydrolase and other enzymes of the leukotriene biosynthetic pathway decreased, consistent with previous reports that TLR-4 agonists do not induce the formation of leukotrienes. Notably, the proteomic data did not always correlate with the transcriptomic data, emphasizing the importance of measuring protein levels and highlighting the existence of mechanisms that operate in addition to transcription to regulate eicosanoid synthesis.
Sabidó et al. subsequently investigated the effect on the eicosanoid-related proteome of priming the cells for ATP-gated-receptor-mediated calcium influx and consequent inflammation. The combined stimulation of macrophages using KLA and ATP induced significant changes, many of which were similar to those reported in response to KLA only, in 19 proteins. In some cases, eicosanoid biosynthetic enzymes that were largely unresponsive to KLA alone showed a considerable increase or decrease in abundance when ATP was added. By contrast, other enzymes were not significantly affected. Notably, however, the double stimulation induced a stronger and more sustained effect on prostaglandin biosynthetic enzymes, such as COX-2.
Overall, when considered together with metabolomic and transcriptomic data generated previously under the same experimental conditions, these proteomic data provide a full picture of the eicosanoid pathway in macrophages, thereby also offering valuable insight into the function of these immune cells.
Katrin Legg- Copyright © 2012 Nature Publishing Group, a division of Macmillan Publishers Limited; used with permission
ORIGINAL RESEARCH PAPER
Sabidó, E. et al. Targeted proteomics of the eicosanoid biosynthetic pathway completes an integrated genomics–proteomics–metabolomics picture of cellular metabolism.
Mol. Cell. Proteomics (23 February 2012). doi:10.1074/mcp.M111.014746