This is a FIRST award application from Dr. Ke-He Ruan. The title of the project is PGI & TXA Synthases: Membrane Anchor Structure/Function. Prostaglandin H2 (PGH2) is converted to Prostaglandin I2 (PGI2) via the catalytic action of Prostaglandin I2 synthase (PGIS). PGI2 is a vasodilator and inhibitor of platelet function. It recently has been employed intravenously for the treatment of pulmonary hypertension. An eicosanoid with actions as a platelet agonist and vasoconstrictor is thromboxane A2 (TXA2). TXA2 also forms from PGH2, via another enzyme, thromboxane A2 synthase (TXAS), a member of the cytochrome P450 superfamily. The structure and function of the N-terminal membrane anchor domains of PGIS and TXAS have not been clearly elucidated. Dr. Ruan has found that TXAS has two separate N-terminal anchor segments and the N- terminus itself faces the cytosol. In contrast, PGIS has 16% sequence identity with TXAS and the hydropathy profile in the N- terminal domain is different. Dr. Ruan suggests that the PGIS membrane anchor structure and the PGIS spatial orientation in relation to the membrane may be different from TXAS. PGH2 is synthesized in the lumen of the endoplasmic reticulum and therefore, the orientation of PGIS and TXAS active sites in relation to the membrane may be important for their conversion of PGH2 into biochemically active eicosanoids with different effects. Other results have shown that the substrate access channel of TXAS faces the ER membrane with the same orientation of the N-terminal membrane anchor region. In this project, attempts will be made to understand how the cytochrome P450 enzymes control eicosanoid biosynthesis. Techniques will include peptidoliposome reconstitution, anti-peptide antibodies which are site- specific, immunocytochemistry, circular dichroism studies, molecular modeling and NMR spectroscopy. The membrane interactions of the N-terminal anchor domains of PGIS and TXAS will be characterized. They will be compared with other cytochrome P450 enzymes in microsomes. The topological arrangement of the catalytic portion of PGIS will be identified in the membrane and it will be compared to that of TXAS. This will also include the determination of the 3D structures of the N-terminal membrane anchor domains of PGIS and TXAS. These will be compared with other microsomal P450s. The residues involved with substrate channel entrance in TXAS and PGIS will be identified. The topological relationship between the substrate channel opening and the ER membrane will be characterized. The experiments should result in the ability to construct a working model for the arrangement of PGIS and comparison with TXAS in the ER membrane. The information should allow for comprehension of similarities and differences between PGIS and TXAS as they relate to the ER membrane and how they coordinate with PGH synthase. Comparisons can then be made between the membrane anchor structure of these two eicosanoid-forming P450s and the anchor structures of other microsomal P450s.
