Membrane Fluidity Is a Key Modulator of Membrane Binding, Insertion, and Activity of 5-Lipoxygenase

Abstract

Mammalian 5-lipoxygenase (5-LO) catalyzes conversion of arachidonic acid to leukotrienes, potent mediators of inflammation and allergy. Upon cell stimulation, 5-LO selectively binds to nuclear membranes and becomes activated, yet the mechanism of recruitment of 5-LO to nuclear membranes and the mode of 5-LO-membrane interactions are poorly understood. Here we show that membrane fluidity is an important determinant of membrane binding strength of 5-LO, penetration into the membrane hydrophobic core, and activity of the enzyme. The membrane binding strength and activity of 5-LO increase with the degree of lipid acyl chain cis-unsaturation and reach a plateau with 1-palmitoyl-2-arachidonolyl-sn-glycero-3-phosphocholine (PAPC). A fraction of tryptophans of 5-LO penetrate into the hydrocarbon region of fluid PAPC membranes, but not into solid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine membranes. Our data lead to a novel concept of membrane binding and activation of 5-LO, suggesting that arachidonic-acid-containing lipids, which are present in nuclear membranes at higher fractions than in other cellular membranes, may facilitate preferential membrane binding and insertion of 5-LO through increased membrane fluidity and may thereby modulate the activity of the enzyme. The data presented in this article and earlier data allow construction of a model for membrane-bound 5-LO, including the angular orientation and membrane insertion of the protein.

Location, Location, Location: Compartmentalization of Early Events in Leukotriene Biosynthesis

“…The only part of 5-LOX demonstrated to penetrate the bilayer is the putative membrane insertion loop that contains Trp-75 in the PLAT domain. This amino acid has been proposed to insert ϳ9 Å deep into the bilayer (53). Thus, 5-LOX is tethered to the membrane with calcium activation, but its substrate is bound deep within the hydrophobic bilayer to the helper protein FLAP.…”

Biosynthesis of leukotriene B4 in human polymorphonuclear leukocytes: regulation by cholesterol and other lipids

“…The nuclear envelope was described as a location site of LT biosynthetic metabolon , with only FLAP integrally associated in the membrane (Woods et al, 1993). Binding of Ca 2+ to 5-LO induces interaction of 5-LO with nuclear membrane with higher affinity to phosphatidylcholine (Puustinen et al, 1988;Kulkarni et al, 2002;Pande et al, 2005). It has been revealed that under physiological conditions phosphatidylcholine content in nuclear membrane is higher than in plasma membrane (Sleight and Pagano, 1984).…”

Impact of Embedded Endocannabinoids and Their Oxygenation by Lipoxygenase on Membrane Properties

“…Membrane insertion of LOX-1 was monitored by FRET experiments, where protein tryptophans served as donors and the Pyrene PE fluorescent probe, incorporated into 1 mM LUVs at a concentration of 10 nM (probe/lipid molar ratio of 1:10 5 ), as a fluorescence acceptor. 35 Fluorescence emission spectra were recorded in the wavelength range from 300 to 600 nm, using an excitation wavelength of 295 nm. The binding isotherms were built as already reported.…”

“…In-depth investigations performed by means of Fourier transformed infrared spectroscopy (FTIR), in both transmission and reflection modes, have shown that membrane binding and enzymatic activity of human 5-LOX increase with the degree of cis-unsaturation of the lipid acyl chain. 35 Conversely, possible effects of LOXs on membrane properties remain largely unknown. In this context, it should be recalled that different animal and plant lipoxygenases (soybean LOX-1 included) have been shown to oxygenate substrates that are embedded within lipid bilayers.…”