The enzyme 5-lipoxygenase (5-LOX) initiates the synthesis of pro-inflammatory leukotrienes. These lipid mediators are synthesized from arachidonic acid (AA) released from the bilayer by the action of Ca2+-dependent phospholipase A2. 5-LOX activity is short-lived, and temporal control appears in part due to an intrinsic instability of the enzyme. This instability provides a mechanism for auto-regulation, preventing an over-production of pro-inflammatory leukotrienes. However, """"""""programmed obsolescence"""""""" is not common to all lipoxygenases, and stable isoforms have been identified. We propose to address three critical aspects of control of 5-LOX activity: (1) Product specificity: The substrate for 5-LOX is the polyunsaturated eicosanoid arachidonic acid. The first step of the reaction is the abstraction of hydrogen from the central carbon of a pentadiene. AA has three pentadiene moieties (and six possible sites of peroxidation, each with either R- or S- chirality). Yet animal lipoxygenases generally produce a single, regio- and stereo- specific product. We will develop a model for 5-LOX specificity that is consistent with its product specificty. We have a 2.86E resolution structure of an engineered 5-LOX that establishes the foundation for these biochemical and structural studies. (2) Programmed obsolescence """"""""Programmed obsolescence"""""""" in 5-LOX appears to have two components: structural instability and turnover-based suicide inhibition. Our data, including our stable mutant form of 5-LOX, suggest that features unique to 5-LOX result in a tenuously restrained C- terminus that contributes to 5-LOX instability. Experiments to define the molecular basis for non- turnover and turnover-based inactivation are proposed. (3) Compartmentalization Ca2+- dependent membrane binding of 5-LOX targets the enzyme to substrate reservoirs and promotes proximity to downstream enzyme activities. Experimental data support a model in which specific Ca2+ binding sites stabilize """"""""insertion loops"""""""" in the C2-like domain of 5-LOX. Others have suggested that 5-LOX binds to its helper protein FLAP, an integral membrane protein. We propose experiments to define the interaction of 5-LOX with the bilayer and determine whether the catalytic domain interacts with the membrane as well, and whether FLAP hands off the substrate to the enzyme, or simply concentrates the AA in the membrane.
Effective therapeutic strategies require that drugs be specific for their protein targets, and therefore the structures of these targets, as well as an understanding of their molecular mechanisms, are essential to guide the development of new medicines. Because of its pivotal role in the biosynthesis of inflammatory leukotrienes, the enzyme 5-lipoxygenase is a target for drugs to treat asthma. The proposed studies will provide both structural and mechanistic information for this key enzyme.
|Gerstmeier, Jana; Newcomer, Marcia E; Dennhardt, Sophie et al. (2016) 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation. FASEB J 30:1892-900|
|Bender, Gunes; Schexnaydre, Erin E; Murphy, Robert C et al. (2016) Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart: IMPLICATIONS FOR MURINE MODELS OF ATHEROSCLEROSIS. J Biol Chem 291:19413-24|
|Mashhadi, Zahra; Newcomer, Marcia E; Brash, Alan R (2016) The Thr-His Connection on the Distal Heme of Catalase-Related Hemoproteins: A Hallmark of Reaction with Fatty Acid Hydroperoxides. Chembiochem 17:2000-2006|
|Mitra, Sunayana; Bartlett, Sue G; Newcomer, Marcia E (2015) Identification of the Substrate Access Portal of 5-Lipoxygenase. Biochemistry 54:6333-42|
|Newcomer, Marcia E; Brash, Alan R (2015) The structural basis for specificity in lipoxygenase catalysis. Protein Sci 24:298-309|
|Kobe, Matthew J; Neau, David B; Mitchell, Caitlin E et al. (2014) The structure of human 15-lipoxygenase-2 with a substrate mimic. J Biol Chem 289:8562-9|
|Lee, Kin Sing Stephen; Liu, Jun-Yan; Wagner, Karen M et al. (2014) Optimized inhibitors of soluble epoxide hydrolase improve in vitro target residence time and in vivo efficacy. J Med Chem 57:7016-30|
|Neau, David B; Bender, Gunes; Boeglin, William E et al. (2014) Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase. J Biol Chem 289:31905-13|
|Morisseau, Christophe; Pakhomova, Svitlana; Hwang, Sung Hee et al. (2013) Inhibition of soluble epoxide hydrolase by fulvestrant and sulfoxides. Bioorg Med Chem Lett 23:3818-21|
|Pecic, Stevan; Pakhomova, Svetlana; Newcomer, Marcia E et al. (2013) Synthesis and structure-activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors. Bioorg Med Chem Lett 23:417-21|
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