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Abstract:
Lipid homeostasis is an essential factor for proper function both at the level of a cell and of an organism. Dysregulation of this process is responsible for some of the major health concerns of our societies such as obesity and atherosclerosis. Considering the wide variety of lipids and the high dynamic of the system, it is clear that regulation is required to keep the appropriate balance between the different lipids. Although a few regulation pathways are already characterized, some others still wait to be discovered. In order to unravel new players involved in lipid regulation, a screening procedure was developed combining RNA interference in Hela cells and thin layer chromatography. Thereby, it is possible to monitor modifications of lipid composition resulting from siRNA knock-down. Furthermore, the potential of mass spectrometry as a lipid analysis tool in large-scale studies was evaluated. This method was then applied to an essential family of regulatory proteins, the kinases. Lipid composition of 600 kinases knock-downs was analyzed. Mostly, variations in triglyceride and cholesterol levels were observed, suggesting that these lipids are more subject to variation in the cells. Unfortunately, it appears that the screen suffered from a high-rate of off-targets effects, implying that most of the phenotypes observed cant reliably be linked to the corresponding kinase knock-downs. However, several interesting conclusions can still be derived from this screen. First, it was observed that several siRNA induce a decrease in cholesterol, which is coupled to accumulation of a new lipid. Several lines of evidence suggest that this new lipid is in fact a methylated sterol precursor such as lanosterol or demethyllanosterol. Previously, HMG-CoA reductase was considered as the rate-limiting enzyme of cholesterol biosynthesis and the major regulated step of this process. These data show that another major regulation step occurs more downstream in the pathway at the level of methylated cholesterol precursors. Furthermore, MAPK9 was identified as a new regulator of triglyceride homeostasis at the cellular level. Upon MAPK9 knock-down, an increase in triglyceride content was observed both by thin layer chromatography and mass spectrometry. Accordingly, these cells present an increase in lipid droplets, the cellular organelles responsible for triglyceride storage. Sty1 was also identified as the functional homolog of MAPK9 in S. pombe for this process, as similar increase in triglyceride and lipid droplets is observed in a deletion strain for this gene. Although more detailed studies will be necessary to unravel the molecular mechanism of this process, these data suggest the evolutionary conserved implication of the MAP kinase pathway in the regulation of lipid storage both in humans and in yeasts.
