? Thromboxane synthase (TXAS) is a """"""""non-classical"""""""" cytochrome P450 that converts prostaglandin H2 to thromboxane A2 (TXA2), a potent inducer of vasoconstriction and platelet aggregation. TXA2 is believed to be a crucial factor contributing to a variety of cardiovascular and pulmonary diseases such as atherosclerosis, myocardial infarction and primary pulmonary hypertension. To understand how the biosynthesis of TXA2 is controlled, it is essential to have an integrated knowledge of TXAS structure/function relationship and its gene regulation. We showed that TXAS shared similar spectral features with other P450s but, unlike other P450s, had a very high turnover number and catalyzed a """"""""peroxidase-like"""""""" reaction with a rate-limiting step at the substrate binding. To continue our efforts in understanding this important enzyme, our specific aims are to (1) evaluate the mono-oxygenase potential of TXAS by determining the redox potential of TXAS heme-iron, characterizing the interaction of TXAS with P450 reductase and investigating the hydroxylase reaction using oxene-donating reagent, (2) determine the structure/function relationship of the active site residues in TXAS catalysis by site-directed mutagenesis and self-inactivation analysis, and (3) solve crystallographic structures of TXAS and its complexes with substrate analog. Regulation of TXAS expression occurs at the transcriptional level. TXAS expression level is cell-type preferential; high in hematopoietic cells and low in non-hematopoietic cells. The binding site for NF-E2 is the most important cis-element for TXAS expression in vivo. The transcription factors p45 NF-E2 and mafG form a heterodimer which activates TXAS transcription in hematopoietic cells. In the non-hematopoietic A549 cells, Nrf2 activated TXAS expression through the NF-E2 site. Thus, TXAS gene utilizes a single cis-acting element but different trans-acting factors to achieve cell-preferential expression. Our results also demonstrated that chromatin structure plays a critical role in TXAS expression. We showed that binding of NF-E2 is associated with the disruption of nucleosomal structure of TXAS promoter. This disruption is likely via CBP/p300. We hypothesize that the differential abilities of NF-E2 proteins in NF-E2 binding, recruiting CBP/p300, disrupting the nucleosomal structure and exerting their trans-activation potency from the TXAS promoter context account for the cell-preferential expression. To test this hypothesis, we propose to: (4) characterize the role of the NF-E2 in altering the nucleosomal structure of TXAS gene and to delineate the TXAS cell-preferential expression using an in vitro chromatin system. ? ?
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