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© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.Kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are two major, closely related receptor subtypes in the glutamate ion channel family. Excessive activities of these receptors have been implicated in a number of central nervous system (CNS) diseases. Designing potent and selective antagonists of these receptors, especially of kainate receptors, is useful for developing potential treatment strategies for these neurological diseases. Here, we report on two RNA aptamers designed to individually inhibit kainate and AMPA receptors. To improve the biostability of these aptamers, we also chemically modified these aptamers by substituting their 2'-OH group with 2'-fluorine. These 2'-fluoro aptamers, FB9s-b and FB9s-r, were markedly resistant to ribonuclease-catalyzed degradation, with a half-life of ~5 days in rat cerebrospinal fluid or serum-containing medium. Furthermore, FB9s-r blocked AMPA receptor activity. Aptamer FB9s-b selectively inhibited GluK1 and GluK2 kainate receptor subunits and also GluK1/GluK5 and GluK2/GluK5 heteromeric kainate receptors with equal potency. This inhibitory profile makes FB9s-b a powerful template for developing tool molecules and drug candidates for treatment of neurological diseases involving excessive activities of the GluK1 and GluK2 subunits. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces the dextran of the highest molar mass reported to date (~109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very high molar mass polymer. In the domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of Glycoside-Hydrolase 70 (GH70) glucansucrases and known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced a quasi-complete loss of very high molar mass dextran synthesis, resulting in the production of only 10-13 kg/mol polymers. Moreover, the double mutation completely switched the semi-processive mode of DSR-OKΔ1 towards a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relatively to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role on the control of processivity. CPI203 A deep structural characterization, if possible with large molar mass sugar ligands, would allow comforting this hypothesis. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 [PNPLA8]), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increasesd under license by The American Society for Biochemistry and Molecular Biology, Inc.Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signalling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP downregulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also downregulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signalling protein extracellular signal-regulated kinase (ERK) and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP downregulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
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