Lanosterol 14alpha-demethylase in biosynthesis of cholesterol and signalling sterols

Disturbed cholesterol homeostasis leads to hyperlipidemias that represent one of the major causes of death in developed societies. The preceeding project contributed importantly to understanding the tissue-specific mechanisms of cholesterol and meiosis activating sterol (MAS) synthesis, at the level of gene regulation (trans-activation of lanosterol 14alpha demethylase CYP51 in sterol-repressed conditions), protein expression (CYP51 enzymatic activity in acrosomal membranes of sperm) and intracellular transport (traffick from ER through the Golgi to the acrosome). The vision of this project is to translate the knowledge of the multi-level regulation of CYP51 to other genes of cholesterol homeostasis (the STEROLTALK group), by applying functional genomic tools in studies of the cross-talk of cholesterol homeostasis with endogenous and exogenous pathways. Due to a high evolutionarily conservation of cholesterol synthesis and metabolism in mammals, several animal models will be applied, from mouse (in vivo studies) to big mammals (bull and ram), in addition to human immortal cell lines. Regulation of cholesterogenic genes will be studied, with particular emphasis on tissue-sepcificity and signalling pathways, that interact with the cholesterol feedback loop. By applying expression profiling by DNA chips and real time PCR and promoter studies, the physiological and patophysiological conditions that allow a cholesterol-feedback independent expression will be identified. The amount of cholesterol and MAS will be measured in animal tissues and cell cultures where different signalling pathways will be activated. Methods for proteome studies will be applied to determine the composition of CYP51 protein compelxes in Golgi and on the acrosome. The CYP51 enzyme activity will be measured in different subcellular fractions and the intracellular transport of CYP51-GFP-fusion protein evaluated by confocal microscopy. Factors that prevent the homologous recombination of the cyp51-lox P constructs into the mouse genome will be determined and new recombination constructs prepared, contributing to the assession of the final goal - a cyp51 conditional knockout mouse by the Cre-loxP technology.