Modulation of human 5-lipoxygenase activity by membrane lipids

Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca(2+)-mediated translocation to membranes, and demonstrates the characteristic features of interfacially activated enzymes, yet the mechanism of membrane binding of 5-LO is not well understood. In an attempt to understand the mechanism of lipid-mediated activation of 5-LO, we have studied the effects of a large set of lipids on human recombinant 5-LO activity, as well as mutual structural effects of 5-LO and membranes. In the presence of 0.35 mM phosphatidylcholine (PC) and 0.2 mM Ca(2+), there was substrate inhibition at >100 microM AA. Data analysis at low AA concentrations yielded the following: K(m) approximately 103 microM and k(cat) approximately 56 s(-1). 5-LO activity was supported by PC more than by any other lipid tested except for a cationic lipid, which was more stimulatory than PC. Binding of 5-LO to zwitterionic and acidic membranes was relatively weak; the extent of binding increased 4-8 times in the presence of Ca(2+), whereas binding to cationic membranes was stronger and essentially Ca(2+)-independent. Polarized attenuated total reflection infrared experiments implied that 5-LO binds to membranes at a defined orientation with the symmetry axis of the putative N-terminal beta-barrel tilted approximately 45 degrees from the membrane normal. Furthermore, membrane binding of 5-LO resulted in dehydration of the membrane surface and was paralleled with stabilization of the structures of both 5-LO and the membrane. Our results provide insight into the understanding of the effects of membrane surface properties on 5-LO-membrane interactions and the interfacial activation of 5-LO.     

Effect of age on reproductive attributes of an aphidophagous ladybird, Cheilomenes sexmaculata

The effect of both male and female age was investigated on certain reproductive attributes, viz. mating incidence, mating duration, fecundity, percent egg viability, ratio of reproductive and non‐reproductive periods and reproductive rate, of an aphidophagous ladybird, Cheilomenes sexmaculata (Fabricius). Females started mating at the age of 8 hours post‐emergence (PE) and males at the age of 2 days PE. Mating in the laboratory was a male‐dominated phenomenon. The mating duration and reproductive rate of 10‐day‐old females when mated with males of varying ages increased up to the male age of 60 days, and thereafter decreased, whereas, fecundity, egg viability and ratio of reproductive and non‐reproductive periods increased up to the male age of 50 days, and thereafter declined. However, when females of varying ages were mated with 10‐day‐old males, fecundity and reproductive rate increased up to 40 days of female age, respectively, then decreased. The ratio of reproductive and non‐reproductive periods increased with increasing age of females. Mating age for optimal reproductive output was 10J50‐day‐old males and NE to 40‐day‐old females. Reproductive cessation in males was recorded after 50 days PE, whereas in females at the age of 40 days PE. Higher mating durations lead to elevated reproductive rates. Delay in the reproductive phase was positively correlated with longevity. The results of this study may aid mass multiplication of this ladybird by identifying and promoting usage of adults of optimal age. Our results also enhance our understanding of the effect of age on reproductive attributes in ladybirds.     

Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.     

Isoform-specific membrane insertion of secretory phospholipase A2 and functional implications

Despite increasing evidence that the membrane-binding mode of interfacial enzymes including the depth of membrane insertion is crucial for their function, the membrane insertion of phospholipase A(2) (PLA(2)) enzymes has not been studied systematically. Here, we analyze the membrane insertion of human group IB PLA(2) (hIBPLA(2)) and compare it with that of a structurally homologous V3W mutant of human group IIA PLA(2) (V3W-hIIAPLA(2)) and with a structurally divergent group III bee venom PLA(2) (bvPLA(2)). Increasing the anionic charge of membranes results in a blue shift of the fluorescence of Trp(3) of hIBPLA(2), a decrease in quenching by acrylamide, and an increase in enzyme activity, reflecting an enhancement in the membrane binding of PLA(2). Fluorescence quenching by brominated lipids indicates significant penetration of Trp(3) into fluid POPC/POPG membranes but little insertion into the solid DPPC/DPPG membranes. Increased membrane fluidity also supports hIBPLA(2) activity, suggesting that membrane insertion of hIBPLA(2) is controlled by membrane fluidity and is necessary for the full activity of the enzyme. Trp fluorescence quenching of the V3W-hIIAPLA(2) and bvPLA(2) by water- and membrane-soluble quenchers indicates substantial membrane insertion of Trp(3) of V3W-hIIAPLA(2), similar to that found for hIBPLA(2), and no insertion of tryptophans of bvPLA(2). Our results provide evidence that (a) structurally similar group IB and IIA PLA(2)s, but not structurally diverse group III PLA(2), significantly penetrate into membranes; (b) membrane insertion is controlled by membrane fluidity and facilitates activation of IB and IIA PLA(2)s; and (c) structurally distinct PLA(2) isoforms may employ different tactics of substrate accession/product release during lipid hydrolysis.     

The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii

One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ.     

Effects of Time-Dependent Stimuli in a Competitive Neural Network Model of Perceptual Rivalry

We analyze a competitive neural network model of perceptual rivalry that receives time-varying inputs. Time-dependence of inputs can be discrete or smooth. Spike frequency adaptation provides negative feedback that generates network oscillations when inputs are constant in time. Oscillations that resemble perceptual rivalry involve only one population being “ON” at a time, which represents the dominance of a single percept at a time. As shown in Laing and Chow (J. Comput. Neurosci. 12(1):39–53, 2002), for sufficiently high contrast, one can derive relationships between dominance times and contrast that agree with Levelt’s propositions (Levelt in On binocular rivalry, 1965). Time-dependent stimuli give rise to novel network oscillations where both, one, or neither populations are “ON” at any given time. When a single population receives an interrupted stimulus, the fundamental mode of behavior we find is phase-locking, where the temporally driven population locks its state to the stimulus. Other behaviors are analyzed as bifurcations from this forced oscillation, using fast/slow analysis that exploits the slow timescale of adaptation. When both populations receive time-varying input, we find mixtures of fusion and sole population dominance, and we partition parameter space into particular oscillation types. Finally, when a single population’s input contrast is smoothly varied in time, 1:n mode-locked states arise through period-adding bifurcations beyond phase-locking. Our results provide several testable predictions for future psychophysical experiments on perceptual rivalry.     

Cyclic AMP increases COX-2 expression via mitogen-activated kinase in human myometrial cells

Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI(2) , acting via cAMP/PKA, was found to increase contraction-associated protein expression in myometrial cells and to promote oxytocin-driven myometrial contractility. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX-2 expression, synthesis and activity. Three cAMP agonists (8-bromo-cAMP, forskolin and rolipram) increased COX-2 mRNA expression and further studies confirmed that this was associated with COX-2 protein synthesis and activity (increased PGE(2) and PGI(2) in culture supernatant) in primary cultures of human myometrial cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP-effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP-effector PDZ-GEF(1-2) , without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE(2) acts through EP-2 to activate MAPK and increase COX-2. These data provide further evidence in support of a dual role for cAMP in the regulation of myometrial function.