Molecular pharmacology of proteins in the postsqualene cholesterol biosynthesis pathway

In contrast to presqualene cholesterol biosynthesis which is of outstanding pharmacological interest and therapeutic relevance, the postsqualene pathway was and still is largely unexplored in mammals, both with respect to biochemistry as well as to, pharmacology. - We biochemically purified, cloned and characterised the mammalian Delta8- Delta7-sterol isomerase (Emopamil Binding Protein, EBP) as a binding protein and putative target for a variety of drugs that exert antiischemic activity in animal models and is defective in the Conradi-Hünermann-Happle Syndrome. - We biochemically purified and cloned the enigmatic sigma1-receptor that is the mammalian homologue of the yeast Delta8- Delta7-sterol isomerase. This protein is expressed in high levels in mammalian brain, whereas the Delta8- Delta7-sterol isomerase (EBP) is less abundant here, suggesting a brain specific function in sterol metabolism of the sigma1-receptor. Sigma1-receptors are of great pharmacological interest since more than two decades but still of unknown function. - We cloned the cDNA and the gene for the mammalian Delta7-sterol reductase (from human and mouse) which, when expressed in heterologous systems is inhibited by the teratogenic drugs (e.g. AY9944). This teratogenic drug target in the postsqualene cholesterol biosynthetic pathway regulates the level of Delta7-dehydroxycholesterol in the skin of mammals and is deficient in the Smith-Lemli-Opitz Syndrome (SLOS). SLOS is an inborn disease of autosomal recessive heredity and an outstanding example for a single genetically determined metabolic deficiency that causes multiple malformations. - Subsequently, we identified mutations in the Delta7-sterol reductase gene in patients with the Smith-Lemli-Opitz Syndrome. - We have now created the first animal model (transgenic mouse) for SLOS. Based on these successes the first aim of the present proposal is to generate sigma1-receptor knock-out mice, to find clues to the biological function of the enigmatic protein and its role in sigma1-ligand pharmacology. The second aim is to characterise the Delta7-sterol reductase knockout model in greater detail to pave the way for future gene therapy. The third aim is to study the enzyme Delta7-sterol reductase as a drug target by developing radioactive and fluorescent labels that allow rapid throughput screening and counterscreening of drugs. The fourth aim is to study the regulation of the enzyme at the genomic and the protein level (e.g. by phosphorylation). The tasks will be performed in collaboration with national and international scientists both in academia and industry. The results are expected to provide significant contributions to the field of postsqualene cholesterol pharmacology and at the interfaces of genetics, biochemistry and clinics.

Our work concentrated on proteins participating in the synthesis of cholesterol (3ß-hydroxysteroid 7-reductase, E.C. 1.3.1.21; 8-7 sterol isomerase, EC 5.3.3.5). As we had purified and cloned the sigma1 receptor which has structural homology to the sterol isomerase of yeast (ERG2), we searched for proteins homologous to the isomerase. ERG2, the 8-7 sterol isomerase and sigma 1 receptors are all high affinity binding proteins for the sigma ligand ifenprodil. For ERG2 and the 8-7 sterol isomerase binding of intermediates of cholesterol biosynthesis is undisputed. However, little is known on sterol binding properties of sigma 1 receptors or proteins related to the isomerase. As a working hypothesis we assume that these proteins serve as binding partners for steroids in signalling pathways or as transporters. In brain e.g. where neuro-steroids are synthesized from cholesterol, the regulation of this pathway is completely unresolved. Proteins such as Hedgehog carry a cholesterol ester at the C-terminus of their signalling domain. Interaction partners on the membrane insertion and, especially the releasing process of such cholesterol-modified proteins are not known. We cloned and expressed a novel human protein homologous to the 8-7 isomerase. This 23.2 KDa protein was termed emopamil-binding-protein (EBP) like (EBPL). EBPL is encoded by four exons on human chromosome 13q14.2 and is distantly related to the 8-7 isomerase (31% identity, 52% similarity). However, EBPL did not bind several tritiated sigma receptor ligands, failed to complement the erg 2-3 mutation in the yeast strain WAO, even when we mutated Trp91 in EBPL to the catalytically essential Tyr in the sterol isomerase (Tyr-111). By blocking sterol biosynthesis in HepG2 cells with mevinolin, the 8-7 isomerase and the 7-sterol reductase mRNAs were upregulated but EBPL mRNA was not. We conclude that EBPLs (like sigma 1 receptors) derived from enzymes of sterol biosynthesis, may have acquired novel functions in evolution. A knock-out mouse model is currently generated. A mouse model for the Smith-Lemli-Opitz Syndrome (SLOS, MIM 270400) was also generated. The mice lack the 7-sterol reductase (obligate step in cholesterol biosynthesis). The k.o. mice did not suckle, breathed with difficulty and died soon after birth. Despite upregulation of mRNA for HMG-CoA - reductase, the levels of activity as well as the half-life of the enzyme was markedly reduced. The null mice offer an excellent model especially in cell culture experiments (with embryonic cells) to study the molecular events that lead to malformations and e.g. synthesis of abnormal steroids and neurosteroids.