Selective regulation of human TRAAK channels by biologically active phospholipids.
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ABSTRACT:
SUBMITTER: Schrecke S
PROVIDER: S-EPMC7746637 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The role of platelet-activating factor (PAF) as a mediator appeared in rather primitive organisms like protozoans and was maintained in more evolved organisms. No reports exist for the presence of PAF or PAF analogues - or even compounds that exhibit PAF-like activity - in cyanobacteria, even though they belong to a a group of organisms at a low evolutionary level where the content of alkylacyl forms of ether lipids is expected to be high. In addition, cyanobacteria serve as a rich source of novel bioactive metabolites. In the present study the total lipids of a strain of Scytonema julianum, a filamentous cyanobacterium isolated from a Greek cave, were separated into neutral lipids and phospholipids, the latter being further fractionated by HPLC. Each phospholipid fraction was tested in vitro for its ability to inhibit PAF-, arachidonic acid- and ADP-induced washed-rabbit-platelet aggregation and/or to cause platelet aggregation. Two types of phospholipids causing platelet aggregation were detected and shown to be an acetylsphingomyelin and an acylacetylglycerol phosphoacetylated glycolipid. The existence of the sphingomyelin analogues is very important, since ceramides, cerebrosides and related lipids are intracellular second messengers. The identification of the phosphoglycoglycerolipid demonstrates a new type of lipid in cyanobacteria, namely one that exhibits a biological activity very similar to that of PAF. Its presence reinforces the concept that PAF is a member of a large family of lipid mediators, apparently having different physiological roles in prokaryotic and eukaryotic organisms. In addition, Scytonema julianum contains a phosphatidylcholine (C(16:0)/(18:2)), even though bacteria in general seldom contain choline-containing phosphoacylglycerols.
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Project description:Ultrasound modulates the electrical activity of excitable cells and offers advantages over other neuromodulatory techniques; for example, it can be noninvasively transmitted through the skull and focused to deep brain regions. However, the fundamental cellular, molecular, and mechanistic bases of ultrasonic neuromodulation are largely unknown. Here, we demonstrate ultrasound activation of the mechanosensitive K+ channel TRAAK with submillisecond kinetics to an extent comparable to canonical mechanical activation. Single-channel recordings reveal a common basis for ultrasonic and mechanical activation with stimulus-graded destabilization of long-duration closures and promotion of full conductance openings. Ultrasonic energy is transduced to TRAAK through the membrane in the absence of other cellular components, likely increasing membrane tension to promote channel opening. We further demonstrate ultrasonic modulation of neuronally expressed TRAAK. These results suggest mechanosensitive channels underlie physiological responses to ultrasound and could serve as sonogenetic actuators for acoustic neuromodulation of genetically targeted cells.
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Project description:We recently characterized the substrate scope of wild-type RebH and proceeded to evolve variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. A substrate walking approach was used to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3-SS, which provides high conversion of tricyclic tryptoline derivatives; and 4-V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable the functionalization of substrates not accepted by wild-type RebH and demonstrates the utility of RebH variants for the site-selective halogenation of biologically active compounds.
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Project description:The biologically active phospholipids Platelet-activating Factor (PAF) and oxidatively truncated phospholipids from chemical oxidation are increased in the circulation of rats subject to the oxidant stress of chronic ethanol ingestion. Potentially, circulating inflammatory and apoptotic phospholipids correlate to physiologic oxidative stress.PAF and the common oxidatively truncated and biologically active phospholipid azelaoyl phosphatidylcholine (Az-PC) were significantly increased in the plasma of older mice, and in male mice. PAF and Az-PC are very rapidly cleared from the circulation, which was unaffected by age or sex. Platelets exposed to Az-PC display phosphatidylserine on their surface, and occlusive platelet carotid arterial thrombosis is enhanced by aging.Biologically active phospholipids vary in the circulation, with the highest levels being found in older, male mice. Turnover of PAF and the biologically active Az-PC are rapid and are invariant with age and sex, so increased production accounts for the increased concentration and flux of both lipids. Platelets are exposed to plasma Az-PC that depolarizes their mitochondria to increase pro-thrombotic phosphatidylserine expression, and occlusive platelet thrombosis is enhanced in aged mice.Oxidatively modified phospholipids are increased in the circulation during common, mild oxidant stresses of aging, or in male compared to female animals. Turnover of these biologically active phospholipids by rapid transport into liver and kidney is unchanged, so circulating levels reflect continuously increased production.
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Project description:Adenosine, a purine nucleoside, is present at high concentrations in tumors, where it contributes to the failure of immune cells to eliminate cancer cells. The mechanisms responsible for the immunosuppressive properties of adenosine are not fully understood. We tested the hypothesis that adenosine's immunosuppressive functions in human T lymphocytes are in part mediated via modulation of ion channels. The activity of T lymphocytes relies on ion channels. KCa3.1 and Kv1.3 channels control cytokine release and, together with TRPM7, regulate T cell motility. Adenosine selectively inhibited KCa3.1, but not Kv1.3 and TRPM7, in activated human T cells. This effect of adenosine was mainly mediated by A2A receptors, as KCa3.1 inhibition was reversed by SCH58261 (selective A2A receptor antagonist), but not by MRS1754 (A2B receptor antagonist), and it was mimicked by the A2A receptor agonist CGS21680. Furthermore, it was mediated by the cAMP/protein kinase A isoform (PKAI) signaling pathway, as adenylyl-cyclase and PKAI inhibition prevented adenosine effect on KCa3.1. The functional implication of the effect of adenosine on KCa3.1 was determined by measuring T cell motility on ICAM-1 surfaces. Adenosine and CGS21680 inhibited T cell migration. Comparable effects were obtained by KCa3.1 blockade with TRAM-34. Furthermore, the effect of adenosine on cell migration was abolished by pre-exposure to TRAM-34. Additionally, adenosine suppresses IL-2 secretion via KCa3.1 inhibition. Our data indicate that adenosine inhibits KCa3.1 in human T cells via A2A receptor and PKAI, thereby resulting in decreased T cell motility and cytokine release. This mechanism is likely to contribute to decreased immune surveillance in solid tumors.
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Project description:Phosphatidylinositol (PtdIns) serves as an integral component of eukaryotic membranes; however, its biosynthesis in apicomplexan parasites remains poorly understood. Here we show that Toxoplasma gondii-a common intracellular pathogen of humans and animals-can import and co-utilize myo-inositol with the endogenous CDP-diacylglycerol to synthesize PtdIns. Equally, the parasite harbors a functional PtdIns synthase (PIS) containing a catalytically-vital CDP-diacylglycerol phosphotransferase motif in the Golgi apparatus. Auxin-induced depletion of PIS abrogated the lytic cycle of T. gondii in human cells due to defects in cell division, gliding motility, invasion, and egress. Isotope labeling of the PIS mutant in conjunction with lipidomics demonstrated de novo synthesis of specific PtdIns species, while revealing the salvage of other lipid species from the host cell. Not least, the mutant showed decline in phosphatidylthreonine, and elevation of selected phosphatidylserine and phosphatidylglycerol species, indicating a rerouting of CDP-diacylglycerol and homeostatic inter-regulation of anionic phospholipids upon knockdown of PIS. In conclusion, strategic allocation of own and host-derived PtdIns species to gratify its metabolic demand features as a notable adaptive trait of T. gondii. Conceivably, the dependence of T. gondii on de novo lipid synthesis and scavenging can be exploited to develop new anti-infectives.
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Project description:Mechanosensitive ion channels underlie neuronal responses to physical forces in the sensation of touch, hearing, and other mechanical stimuli. The fundamental basis of force transduction in eukaryotic mechanosensitive ion channels is unknown. Are mechanical forces transmitted directly from membrane to channel as in prokaryotic mechanosensors or are they mediated through macromolecular tethers attached to the channel? Here we show in cells that the K(+) channel TRAAK (K2P4.1) is responsive to mechanical forces similar to the ion channel Piezo1 and that mechanical activation of TRAAK can electrically counter Piezo1 activation. We then show that the biophysical origins of force transduction in TRAAK and TREK1 (K2P2.1) two-pore domain K(+) (K2P) channels come from the lipid membrane, not from attached tethers. These findings extend the "force-from-lipid" principle established for prokaryotic mechanosensitive channels MscL and MscS to these eukaryotic mechanosensitive K(+) channels.
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Project description:Inhibitory potassium channels of the TREK1/TRAAK family are integrators of multiple stimuli, including temperature, membrane stretch, polyunsaturated fatty acids and pH. How these signals affect the gating of these channels is the subject of intense research. We have previously identified a cytoplasmic domain, pCt, which plays a major role in controlling channel activity. Here, we use pharmacology to show that the effects of pCt, arachidonic acid, and extracellular pH converge to the same gate within the channel. Using a state-dependent inhibitor, fluoxetine, as well as natural and synthetic openers, we provide further evidence that the "up" and "down" conformations identified by crystallography do not correspond to open and closed states of these channels.
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Project description:BackgroundBifidobacterium spp. are representative probiotics that play an important role in the health of their hosts. Among various Bifidobacterium spp., B. bifidum BGN4 exhibits relatively high cell adhesion to colonic cells and has been reported to have various in vivo and in vitro bio functionalities (e.g., anti-allergic effect, anti-cancer effect, and modulatory effects on immune cells). Interleukin-10 (IL-10) has emerged as a major suppressor of immune response in macrophages and other antigen presenting cells and plays an essential role in the regulation and resolution of inflammation. In this study, recombinant B. bifidum BGN4 [pBESIL10] was developed to deliver human IL-10 effectively to the intestines.ResultsThe vector pBESIL10 was constructed by cloning the human IL-10 gene under a gap promoter and signal peptide from Bifidobacterium spp. into the E. coli-Bifidobacterium shuttle vector pBES2. The secreted human IL-10 from B. bifidum BGN4 [pBESIL10] was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western Blotting, and enzyme-linked immunosorbent assay (ELISA). More than 1,473?±?300 ng/mL (n?=?4) of human IL-10 was obtained in the cell free culture supernatant of B. bifidum BGN4 [pBESIL10]. This productivity is significantly higher than other previously reported human IL-10 level from food grade bacteria. In vitro functional evaluation of the cell free culture supernatant of B. bifidum BGN4 [pBESIL10] revealed significantly inhibited interleukin-6 (IL-6) production in lipopolysaccharide (LPS)-induced Raw 264.7 cells (n?=?6, p?<?0.0001) and interleukin-8 (IL-8) production in LPS-induced HT-29 cells (n?=?6, p?<?0.01) or TNF?-induced HT-29 cells (n?=?6, p?<?0.001).ConclusionB. bifidum BGN4 [pBESIL10] efficiently produces and secretes significant amounts of biologically active human IL-10. The human IL-10 production level in this study is the highest of all human IL-10 production reported to date. Further research should be pursued to evaluate B. bifidum BGN4 [pBESIL10] producing IL-10 as a treatment for various inflammation-related diseases, including inflammatory bowel disease, rheumatoid arthritis, allergic asthma, and cancer immunotherapy.
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