Site-directed mutagenesis studies of h 15-LOX-1 and their effects on inhibitor potency

1. Site-directed mutagenesis studies of h15-LOX-1 and their effects on inhibitor potency Michelle Armstrong, Thomas Horn, Josh Deschamps, Chris van Hoorebeke, Cody Freedman, Theodore Holman Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA. Mammalian lipoxygenases have been linked to inflammation, immune response, cardiovascular disease, asthma, different cancers, arthritis and stroke. Human reticulocyte 15-lipoxygenase-1, which catalyzes the dioxygenation of various polyunsaturated fatty acids, has been implicated in all of these important human diseases and is therefore a relevant target for investigation. Molecular modeling studies of human 15lipoxygenase-1, based on the X-ray crystal structure of rabbit lipoxygenase, suggest specific residues in the active site that may be critical for inhibitor binding. We have generated six mutants (R402L, R404L, F414I, F414W, E356Q, Q547L) of h15-LOX-1 through site-directed mutagenesis to test this model. Steady state kinetic experiments confirmed that the mutations did not affect the catalytic activity of the isozymes. To test whether the active site mutations altered inhibitor binding, 3-pt IC50 screens were conducted on all mutants against two previously published potent and selective h15-LOX-1 inhibitors varying in length: ML351 (short) and an ML094 derivative (long). The steady state inhibition kinetics were investigated only on the mutants that exhibited significant differences relative to wild type h15-LOX-1 in their IC50 potencies. The mutations that affected inhibitor potency relative to wild type h15-LOX-1 were F414I, F414W and E356Q for both inhibitors, ML351 and ML094 derivative. The data implies that both inhibitors interact with the same residues in the active site. Structure activity relationships will be presented. 2. A lipid-mediated proinflammatory response to ionizing radiation Evagelia C. Laiakis 1 , Katrin Strassburg 2 , Steven Lai 3 , Rob J. Vreeken 2 , Thomas Hankemeier 2 , Rob Plumb 3 , Albert J. Fornace Jr. 1 , Giuseppe Astarita 1,3 1 Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC; 2 Netherlands Metabolomics Centre, Leiden University, Leiden, the Netherlands; 3 Waters Corporation, Health Sciences, Milford, MA. Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The deleterious consequences of ionizing radiation exposure can result in cancer and non-cancer-related diseases, including cardiovascular diseases and cognitive decline. The molecular response to ionizing radiation, however, is still not completely understood. Here we screened mouse serum for metabolic alterations following an acute exposure to gamma radiation using a multi-platform, mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and low-abundance, oxygenated, polyunsaturated, fatty acids (PUFAs) in the two groups of animals. Exposure to gamma radiation induced a significant increase in the serum levels of ether phosphatidylcholines (PCs) while decreasing the levels of diacyl PCs carrying PUFAs. In exposed mice, levels of pro-inflammatory, oxygenated metabolites of arachidonic acid increased, whereas levels of anti-inflammatory metabolites of omega-3 PUFAs decreased. Our results indicate a specific serum lipidomic biosignature, which could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine. For all poster abstracts: Presenting author is underlined # indicates equal effort contributed by first authors * indicates lightning talk selection 3. Supercritical fluid chromatography-mass spectrometry approaches for lipidomics profiling Anne E. Blackwell, Joseph L. Hedrick Agilent Technologies, Wilmington, DE. Given the complexity of biological matrices, long chromatography is often needed for lipidomics and metabolomics profiling experiments, resulting in extremely low throughput. When combined with the large numbers of samples needed for good statistics, profiling is time consuming and expensive. The use of supercriticial fluid chromatography (SFC) offers significantly shorter runs than liquid chromatography while preserving or improving chromatographic separation for certain classes of lipids. An SFC coupled to a QTOF mass spectrometer, with full flow sent from the SFC to the ionization source, was used for this work. Complementary chromatographic approaches were investigated. Samples analyzed include the lipid fractions of plasma and liver, cooking oils, and numerous pure chemical standards. SFC-MS was found to be an excellent platform for lipidomics profiling experiments. The chromatographic reproducibility was good, and the approach had several unique strengths, the most obvious of which was speed. Triglycerides (TG) are a lipid class where SFC is particularly advantageous. A mixture of five TG standards (C:8-C:16) were analyzed in liver matrix by reverse-phase C18 LC and C18 SFC with a CO2-MeOH gradient. Using a 38 minute H2O-IPA LC gradient, the five TG standards elute over a 35 minute period. With a 13 minute SFC method, the same standards elute completely resolved within 5 minutes. Using the same two LC and SFC methods, liver lipids were annotated using the METLIN database. The majority of TGs eluted at high IPA in a 2 minute window by LC. With the 13 minute SFC method, annotated TGs elute over a 10 minute window – an improvement in both separation and speed. SFC with a C18 column shows excellent separation of TGs based on chain length and degrees of saturation, giving baseline separation of TGs that differ by a single double bond. Trends for other lipid classes will also be discussed. 4. Untargeted lipidomics reveals loss of key phospholipids and several lysophospholipids from vitamin E-deficient zebrafish brains * Jaewoo Choi 1,2 , Scott W. Leonard 1 , Katherine Kasper 1,4 , Melissa McDougall 1,4 , Jan F. Stevens 1,2,5 , Robert L. Tanguay 3,5 , Maret G. Traber *1,4,5 1 Linus Pauling Institute; 2 College of Pharmacy; 3 Environmental and Molecular Toxicology; 4 College of Public Health and Human Sciences; 5 Environmental Health Sciences Center; Oregon State University, Corvallis, OR. We hypothesized that brains from vitamin E-deficient (E-) zebrafish (Danio rerio) would undergo increased lipid peroxidation and loss of specific, highly polyunsaturated fatty acids. To test this hypothesis, brains from zebrafish fed for 9 months defined diets without (E-) or with added vitamin E (E+, 500 mg RRR-α-tocopheryl acetate/kg diet) were studied. Using an untargeted lipidomic analysis (MS/MSALL), 1-hexadecanoyl-2docosahexaenoyl-sn-glycero-3-phosphocholine (DHA-PC 38:6, PC 16:0/22:6) was the lipid that showed the most significant and greatest fold-differences between groups. Using LC-MS/MS for quantification, we found that DHA-PC 38:6 concentrations in extracts from dissected brains were lower in E(4.3 ± 0.6 μg/mg brain, mean ± SEM) compared with E+ fish (6.5 ± 0.9, P=0.0413). Using UPLC-TOF-MS/MS and lipidomics, 155 lipids in brain extracts were identified. Several lipid species, including four phospholipids (PLs) containing DHA, were lower (p < 0.05) in Ezebrafish brains. Notably, DHA-PC 38:6 had the largest intensities of any of the lipids that were significantly different between groups. Moreover, hydroxy-DHA-PC was increased in Ebrains (P=0.0341) supporting the hypothesis of greater DHA peroxidation. More striking was the depletion in Ebrains of nearly 60% of 19 different lyso-PL (combined P=0.0003), which are critical for membrane PL remodeling. Thus, Ebrains contained less DHA-PL, more hydroxy-DHA-PC and fewer lyso-PLs, suggesting that lipid peroxidation depletes membrane DHA-PC and homeostatic mechanisms to repair the damage result in lyso-PL depletion. DHA-PC 38:6 is a plasma biomarker of increased Alzheimer’s disease risk in humans. Thus, inadequate vitamin E status in zebrafish results in a specific depletion in a critical brain PL that is associated with increased Alzheimer’s disease risk in humans. 5. Elucidating the metabolism of omega-3 and omega-6 endocannabinoids by cardiac CYP2J2 epoxygenase in Nanodiscs * Daniel R. McDougle †,1 , William R. Arnold ‡ , Aditi Das †, ‡,2 † Department of Comparative Biosciences; ‡ Department of Biochemistry; 1 Medical Scholars Program; 2 Beckman Institute for Advanced Science and Technology and Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL. The human body contains endocannabinoids that elicit similar effects as Δ9-tetrahydrocanabinol (THC), the principal component of cannabis, which produces similar psychoactive and anti-nociceptive effects. The two most well studied endocannabinoids include anandamide (AEA) and 2-arachidonoylglycerol (2-AG), which are derived from the omega-6 arachidonic acid (AA). Previously, we showed that both of these omega-6 endocannabinoids are substrates for metabolism by human heart cytochrome P450 2J2 epoxygenase (CYP2J2) to form novel AEA and 2-AG epoxides with distinct biological activity. Recently, the structural diversity of the endocannabinoid family has expanded with the discovery that the omega-3 fatty acids eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) also form endocannabinoids eicoasapentaenoic ethanolamide (EPEA) and docosahexaenoic ethanolamide (DHEA), respectively. Using LC-MS we demonstrate that EPEA and DHEA are metabolized by the epoxygenases such as CYP2J2 to produce novel EPEA and DHEA epoxides with unknown biological functions. In order to accomplish the detailed mechanistic studies, we use th