Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs

Abstract

Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. The enzymes associate with one leaflet of the membrane bilayer. We utilized nanodisc technology to investigate the function of human (hu) COX-2 and murine (mu) COX-2 in a lipid bilayer environment. huCOX-2 and muCOX-2 were incorporated into nanodiscs composed of POPC, POPS, DOPC, or DOPS phospholipids. Size-exclusion chromatography and negative stain electron microscopy confirm that a single COX-2 homodimer is incorporated into the nanodisc scaffold. Nanodisc-reconstituted COX-2 exhibited similar kinetic profiles for the oxygenation of AA, eicosapentaenoic acid, and 1-arachidonoyl glycerol compared to those derived using detergent solubilized enzyme. Moreover, changing the phospholipid composition of the nanodisc did not alter the ability of COX-2 to oxygenate AA or to be inhibited by various nonselective NSAIDs or celecoxib. The cyclooxygenase activity of nanodisc-reconstituted COX-2 was reduced by aspirin acetylation and potentiated by the nonsubstrate fatty acid palmitic acid to the same extent as detergent solubilized enzyme, independent of phospholipid composition. The stabilization and maintenance of activity afforded by the incorporation of the enzyme into nanodiscs generates a native-like lipid bilayer environment to pursue studies of COX utilizing solution-based techniques that are otherwise not tractable in the presence of detergents.

The structure of ibuprofen bound to cyclooxygenase-2

“…Wild-type (WT), R120A, and Y355F constructs were incubated with 100μM ( R/ S)-IBP on ice for 30 minutes before injection into a reaction cuvette containing 100μM AA and 100μM ( R/ S)-IBP. IBP resulted in a 40% decrease in cyclooxygenase activity with WT muCOX-2, consistent with previous studies (Dong et al, 2011; Orlando et al, 2014). Conversely, neither the R120A nor the Y355F constructs were inhibited by ( R/ S)-IBP (Figure 2).…”

“…Cyclooxygenase activity was determined using an oxygen electrode and 100μM arachidonic acid as the substrate as described in (Vecchio et al, 2012). Inhibition studies using IBP were carried out as described in (Orlando et al, 2014). Thermal shift assays were carried out with apo muCOX-2 using a Stratagene Mx3005P real-time PCR instrument as described in (Koszelak-Rosenblum et al, 2008).…”

Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry

“…The S530T/G533V double mutant was generated using G533V muCOX-2 in pFastBac-1 as the template for mutagenesis. His 6 N580A human (hu) COX-2 in pFastBac-1 was used to engineer FLAG huCOX-2 using the protocol described in 30 . The resulting FLAG huCOX-2 construct then served as a template for the deletion of residues 586–612 (Δ586) at the C-terminus 31 .…”

“…The detergent concentration in each buffer after size-exclusion chromatography was 0.53% (w/v) βOG, 0.1% (w/v) Tween-20, 0.1% (w/v) C 10 E 6 , or 0.5% (w/v) CHAPS. The FLAG huCOX-2 construct was used to produce purified protein in βOG and nanodiscs utilizing the protocols outlined in 30 . After the affinity chromatography step, half of the sample was used to proceed through the size-exclusion chromatography step described above 7 .…”

Endocannabinoids Anandamide and 2-Arachidonoylglycerol Are Substrates for Human CYP2J2 Epoxygenase

“…For all studies with recombinant protein, we used the nanoscale lipid bilayers of nanodiscs (ND) to solubilize and stabilize membrane-bound CYP2J2. These model membranes have been previously used to functionally stabilize membrane protein in solution and on surfaces (Nath et al, 2007;Bayburt and Sligar, 2010;Denisov and Sligar, 2011;Das et al, 2014;Orlando et al, 2014). Importantly, the use of these model membranes significantly reduced scattering and enabled spectroscopic characterization by increasing the solubility of the hydrophobic lipid substrates and membrane proteins.…”