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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL056712-02
Application #
2460216
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1996-08-01
Project End
2000-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
Lin, Haocheng; Yuan, Jiuhong; Ruan, Ke-He et al. (2013) COX-2-10aa-PGIS gene therapy improves erectile function in rats after cavernous nerve injury. J Sex Med 10:1476-87
Ruan, Ke-He; Mohite, Anita; So, Shui-Ping et al. (2013) Establishing novel prostacyclin-synthesizing cells with therapeutic potential against heart diseases. Int J Cardiol 163:163-9
Chillar, Annirudha; So, Shui-Ping; Ruan, Cheng-Huai et al. (2011) A profile of NSAID-targeted arachidonic acid metabolisms in human embryonic stem cells (hESCs): implication of the negative effects of NSAIDs on heart tissue regeneration. Int J Cardiol 150:253-9
Ruan, Cheng-Huai; So, Shui-Ping; Ruan, Ke-He (2011) Inducible COX-2 dominates over COX-1 in prostacyclin biosynthesis: mechanisms of COX-2 inhibitor risk to heart disease. Life Sci 88:24-30
Mohite, Anita; Chillar, Annirudha; So, Shui-Ping et al. (2011) Novel mechanism of the vascular protector prostacyclin: regulating microRNA expression. Biochemistry 50:1691-9
Chillar, Annirudha J; Karimi, Parastoo; Tang, Kathy et al. (2011) An agonist sensitive, quick and simple cell-based signaling assay for determination of ligands mimicking prostaglandin E2 or E1 activity through subtype EP1 receptor: Suitable for high throughput screening. BMC Complement Altern Med 11:11
Chillar, Annirudha; Wu, Jiaxin; Cervantes, Vanessa et al. (2010) Structural and functional analysis of the C-terminus of Galphaq in complex with the human thromboxane A2 receptor provides evidence of constitutive activity. Biochemistry 49:6365-74
Ruan, Ke-He; Cervantes, Vanessa; Wu, Jiaxin (2009) Ligand-specific conformation determines agonist activation and antagonist blockade in purified human thromboxane A2 receptor. Biochemistry 48:3157-65
Ruan, Ke-He; Cervantes, Vanessa; So, Shui-Ping (2009) Engineering of a novel hybrid enzyme: an anti-inflammatory drug target with triple catalytic activities directly converting arachidonic acid into the inflammatory prostaglandin E2. Protein Eng Des Sel 22:733-40