Analysis of sterol isomerase related proteins

Two structurally unrelated human genes, sigma1 receptor and EBPL have separately evolved during eukaryotic evolution from ERG2 and EBP, two integral membrane proteins proteins with identical roles in sterol biosynthesis (sterol delta8 delta7 isomerases, EC5.3.3.5). The sigma1 receptor has been linked to a variety of drug effects e.g. protection from cerebral ischemia and is considered a putative therapeutic target. In sigma1 receptor and EBPL the original catalytic activity was lost and enigmatic novel biochemical functions were acquired. Nevertheless for unknown reasons other essential enzyme features such as high affinity ligand binding and amino acid residues required for catalysis were preserved over several hundred millions years of evolution. The aim of the proposal is to elucidate (1) the biological function and putative medical significance of these sterol delta8 delta7 isomerase related proteins by conditional gene disruption in mice, (2) to establish scalable overexpression and purification of the sigma1 receptor and its homologue ERG2 for structural characterization by solid state NMR spectroscopy to understand the relationship between structure, catalysis and high affinity drug binding and (3) to identify sigma1 receptor interacting proteins to link the sigma1 receptor to other cellular processes. These aims also pave the way to determinate the structure of more complex integral membrane proteins e.g. the sterol delta7 reductase (DHCR7). Understanding of the mechanism of drug binding to enzymes of sterol metabolism such as ERG2 might lead to development of conformational stabilizers ("pharmacological chaperones") for mutated enzymes of sterol metabolism.