Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Exhibit Hall A
Paris Elyse Taylor - Louisiana State University
Co-Author(s): Nathaniel C. Gilbert, Ph.D., Louisiana State Univesity, Baton Rouge, LA; Marcia E. Newcomer, Ph.D., Louisiana STate University, Baton Rouge, LA
Heart disease is the No. 1 killer for all Americans. The main focus of my research is to aid in new targeted therapies that interrupt the formation of oxidized low-density lipoprotein particles that cause heart disease. A critical component in the development of atherosclerosis is the transformation of macrophages to foam cells, a process involving the uptake of lipids carried by low-density lipoprotein LDL. Recent studies have revealed that 15-lipoxygenase-2 (15-LOX-2), the major lipoxygenase expressed in macrophages, is found in high abundance in atherosclerotic plaque and is induced by hypoxia and other inflammation factors. The enzyme has been linked to elevated levels of oxidized lipids and is able to directly oxidize polyunsaturated fatty acids esterified in phospholipids. This study is aimed toward the engineering of a mutant form of 15-LOX-2 that will serve as a robust framework for future structural studies. Unfortunately, the wild type enzyme is not suitable for these studies because it crystallizes in such a way that we can?t trap inhibitors in the active site. Mutations will be made that facilitate crystallization, but do not change the overall characteristics of the binding site. To achieve this goal, we removed a loop from the structure of the enzyme that limited the quality of the crystals that can be made with this protein. This mutant 15-LOX-2 LM was expressed in E. coli Rosetta 2 (DE3) cells. The protein was purified by immobilized-metal affinity chromatography followed by size-exclusion chromatography. Monodispersed monomers of 15-LOX-2 LM incubated with inhibitor #356800 was sent to the high-throughput crystallization screening center at Hauptman-Woodward Medical Research Institute in Buffalo, New York. After analyzing the results from HWI, the Hampton Research PEGRx crystal screen was set up in house to replicate conditions seen at HWI. New crystals will be screened at Protein Crystallography beamline at the Center for Advanced Microstructures and Devices. These crystal structures can provide structure-activity relationships between the small molecule and protein, which will aid in new inhibitor design for targeting 15-LOX-2.
Funder Acknowledgement(s): LSU IMSD Program (NIH 2R25GM069743); LSU Office of Research & Economic Development
Faculty Advisor: Nathaniel C. Gilbert, Ph.D., firstname.lastname@example.org
Role: For this research, I assisted with the expression and purification of the 15-LOX-2 LM protein. My biggest contribution comes from setting up all of the different crystal trays and observing them for any signs of crystal formation. I also assist my mentor in determining new crystal screening conditions.