The effects of rosuvastatin and the CYP51A1 inhibitor LEK-935 on the proteome of primary human hepatocytes.

Elevated amounts of cholesterol are thought to be involved in several severe diseases. Despite the fact that many studies have been performed and published, the action of cholesterol-lowering agents used to diminish the plasma cholesterol level is not fully understood yet. In this study, the effects of the HMG-CoA reductase inhibitor rosuvastatin and the new CYP51A1 inhibitor 2-((3,4-dichlorophenethyl)(propyl)amino)-1-(pyridin-3-yl)ethanol (LEK-935) on the proteome of primary human hepatocytes were analyzed for the first time. To get an idea about interindividual differences, two different human donors were used. The cytosolic and microsomal fractions of the cells were analyzed in a semiquantitative manner by two-dimensional-polyacrylamide gel electrophoresis and capillary high-performance liquid chromatography-mass spectrometry, respectively. Thereby, a set of 44 proteins was found to be differentially presented. The chosen experimental set-up was validated by proteins already known to be affected by statins and involved in the cholesterol biosynthesis. Other proteins found to be regulated cannot be directly related to cholesterol metabolism and have not been described to be affected by cholesterol-lowering agents so far. Some of these proteins may represent interesting targets for further investigations into the analysis of severe side-effects as well as pleiotropic effects of the statins. During the proteome analysis of the two different donors, interindividual differences were observed that were validated by real-time reverse transcription-polymerase chain reaction measurements. Thus, new information and a deeper insight into the processes taking place inside cells treated with cholesterol-lowering agents can be drawn from this study.

Shotgun proteomic analysis of the microsomal fraction of eukaryotic cells using a two-dimensional reversed-phase x ion-pair reversed-phase HPLC setup.

A RPxIP-RP HPLC separation scheme was combined with on-line ESI-IT tandem MS or off-line MALDI tandem TOF MS and applied to the analysis of eukaryotic subcellular proteomes. Previous proteomic studies [1] were complemented by the approval of the approach to eukaryotic proteomes using the fission yeast Schizosaccharomyces pombe. The major focus was set to the analysis of primary human hepatocyte microsomes, representing a compartment of high interest due to its involvement in xenobiotic detoxification and cholesterol homeostasis. Of the 588 proteins identified from two donors, 24% are involved in cholesterol homeostasis or xenobiotic/lipid metabolism. Up to 50% of the identified proteins belong to the group of membrane proteins, difficult to investigate using gel-based proteomic approaches. We further demonstrated the reproducibility and comparability of the approach and reduced the amount of sample load by almost 70% with only minor loss of information about the proteins identified in the samples. The presented study clearly demonstrates the good applicability of the experimental setup to the analysis of subcellular proteomes including large membrane fractions, where only low amounts of sample material are available.