Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes

The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.     

Hydrolysis of a Lipid Membrane by Single Enzyme Molecules: Accurate Determination of Kinetic Parameters

The accurate determination of the maximum turnover number and Michaelis constant for membrane enzymes remains challenging. Here, this problem has been solved by observing in parallel the hydrolysis of thousands of individual fluorescently labeled immobilized liposomes each processed by a single phospholipase A2 molecule. The release of the reaction product was tracked using total internal reflection fluorescence microscopy. A statistical analysis of the hydrolysis kinetics was shown to provide the Michaelis–Menten parameters with an accuracy better than 20 % without variation of the initial substrate concentration. The combined single‐liposome and single‐enzyme mode of operation made it also possible to unravel a significant nanoscale dependence of these parameters on membrane curvature.     

Negative feedback regulation of Wnt signaling by G�¦�-mediated reduction of Dishevelled

Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gβ₂) bound to Axin and Gβ₂ inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gβ₂γ₂ mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gβ₂γ₂ into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by β-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gβ_2γ2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gβ_2γ2 is required because a mutant of Gβ₂, Gβ₂ (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gβ₁ and Gβ₂. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled→Gβγ→PLC→Ca⁺²/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gβ₂γ₂ inhibits Wnt signaling by degradation of Dishevelled.     

Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS(-/-) mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor N(G)-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca(2+) levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca(2+) levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol- treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca(2+) chelator, calmodulin antagonist, and CaMKKβ siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca(2+)-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca(2+)/CaMKKβ-dependent eNOS phosphorylation and Ca(2+)-dependent eNOS dimerization.