University of Houston System
Grant: $24,969 - National Institutes of Health - Jun. 1, 2009
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Award Description: The goal of this project is to structurally understand how the thromboxane A2 synthase (TXAS), coordinates (couples) with the upstream cyclooxygenase (COX) isoform-1 (COX-1) vs. isoform-2 (COX-2) in the biosynthesis of TXA2 (a key pro-thrombotic mediator that causes stroke and heart attack). Our innovatively engineered hybrid enzymes that linked the COX-1 (or -2) C-terminus to prostacyclin (PGI2) synthase (PGIS) by a designed linker were able to mimic the coupling reactions of the native COXs with PGIS in the cells. Based on this information, we hypothesize that the other COX downstream synthase, TXAS, has similar mechanisms in coordinating with the COXs to synthesize TXA2. However, it is known that TXAS favors coupling to COX-1 rather than COX-2, yet little information is available for the selectivity. This project is id focused on creating novel hybrid enzymes, linking COX-1 to TXAS and linking COX-2 to TXAS, as models to reveal the mechanisms of the selective coupling. The specific aims are to: 1). Clone an engineered hybrid enzyme linking COX-1 to TXAS by a TM linker and identify its biological activity by overexpressing the hybrid enzyme in mammalian cells, and 2). Clone an engineered hybrid enzyme linking COX-2 to TXAS by a TM liker and identify its activity by overexpressing the enzyme in mammalian cells. The engineered COX-1-linker-TXAS and COX-2-linker-TXAS hybrid enzymes will be used as models mimicking the coordination of the COX-1 and COX-2 with TXAS in the specific biosynthesis of TXA2 from the common substrate, AA, in the cells. The study will provide important insight for understanding the mechanisms involved in controlling the biosynthesis of TXA2 through the COX-1 vs. COX-2 pathways and the design of the next-generation therapeutic strategies against thrombosis and stroke. One educator from a local high school will be recruited to participate in the studies under the PI’s supervision. The short term benefit will be the accomplishment of the aims and the training for the participant. The long term benefit will be that the participant would influence and encourage her/his students and colleagues to pursue research careers in health-related sciences.
Project Description: Specific Aims: Aim 1) Engineering a functional hybrid enzyme linking the C-terminus of human COX-1 to the N-terminus of human TXAS (COX-linker-TXAS) to mimic the COX-1’s coordination with TXAS in the ER membrane. Aim 2) Engineering a functional hybrid enzyme linking the C-terminus of human COX-2 to the N-terminus of human TXAS (COX-2-linker-TXAS) to mimic the COX-2’s coordination with TXAS in the ER membrane. Studies and Results: A). Hybrid enzyme cDNA cloning - The hybrid enzymes of COX-1 or -2 with TXAS were generated by general PCR approaches. Briefly, the corresponding synthase cDNA was isolated from the vector (pcDNA3.1) by PCR using primers containing the cutting sites. The resultant cDNA segment was cut with the corresponding enzyme and ligated into the corresponding sites in the vector. B). Expression of the recombinant enzymes in cells - The engineered hybrid enzymes were expressed in COS-7 and HEK293 cells. Briefly, the cells were grown for 24 hrs, then transected with a purified cDNA by Lipofectamine (Invitrogen) or other transfection reagents. Approximately 48 hrs post-transfection, some cells were harvested for further activity and protein analyses, and the remainder were used for preparation of the stable expression cell lines by G418 screening. For the co-transfection, different ratios of the two cDNAs were used. C). Coupling assays - The catalytic enzymatic reactions involved in the conversion of AA into the end-products, PGI2, TXA2, and/or mPGES-1 begin with the addition of [14C]-AA into the sample containing the enzymes. After incubation for varying times, the reactions were stopped with 0.1% acetic acid containing 35% acetonitrile. The end-product profile, including [14C]-TXB2 (degraded [14C]-TXA2) is separated by a C18 analytical column using an HPLC connected to a flow scintillation analyzer to form an isotope-HPLC analysis system. LC/MS was used to analyze the produced prostanoids following the addition of unlabeled AA and endogenous AA.
Infrastructure Description: n/a
Jobs Summary: The University of Houston values research as one of its top institutional priorities; it is a vital part of the university’s strategic vision. To take our place among the nation’s great metropolitan research institutions, we are building a well-rounded, robust research core group , made up of outstanding faculty and staff. This core group will ensure production of innovative research important to our community and beyond. The ARRA funding is critical to the building of that research core group. The workforce for ARRA funded research projects may include academic faculty and staff with the following titles: Principal Investigator, Co-Principal Investigator, Professor, Associate Professor, Assistant Professor, Research Scientist, Research Professor, Research Associate Professor, Research Assistant Professor, Senior Research Scientist, Graduate Research Assistant, Research Associate, Lab Technician, Research Lab Manager, Post Doctoral Fellow. Each of these titles contributes to the future of discovery — scientific and otherwise. (Total jobs reported: 0)
Project Status: Less Than 50% Completed
This award's data was last updated on Jun. 1, 2009. Help expand these official descriptions using the wiki below.
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