scholarly journals Regulation of Bacillus subtilis DesK thermosensor by lipids

2013 ◽  
Vol 451 (2) ◽  
pp. 269-275 ◽  
Author(s):  
Mariana Martín ◽  
Diego de Mendoza
Keyword(s):  
Bacillus Subtilis ◽  
Lipid Composition ◽  
Group Structure ◽  
Acyl Chain ◽  
Kinase Domain ◽  
Head Group ◽  
Fatty Acyl Chain ◽  
Synthetic Approach ◽  
Membrane Thickness

Temperature sensing is essential for the survival of living cells. The membrane-bound thermosensor DesK from Bacillus subtilis is a key representative of histidine kinases receptors able to remodel membrane lipid composition when the temperature drops below ~30°C. Although the receptor is well studied, a central issue remains: how does the compositional and functional diversity of the surrounding membrane modulate receptor function? Reconstituting full-length DesK into proteoliposomes of well-defined and controlled lipid composition represents a minimal synthetic approach to systematically address this question. Thus DesK has been reconstituted in a variety of phospholipid bilayers and its temperature-regulated autokinase activity determined as function of fatty acyl chain length, lipid head-group structure and phase preference. We show that the head group structure of lipids (both in vitro and in vivo) has little effect on DesK thermosensing, whereas properties determined by the acyl chain of lipids, such as membrane thickness and phase separation into coexisting lipid domains, exert a profound regulatory effect on kinase domain activation at low temperatures. These experiments suggest that the non-polar domain of glycerolipids is essential to regulate the allosteric structural transitions of DesK, by activating the autophosphorylation of the intracellular kinase domain in response to a decrease in temperature.

scholarly journals Profile of Phosphatidylserine Modifications under Nitroxidative Stress Conditions Using a Liquid Chromatography-Mass Spectrometry Based Approach

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 107 ◽  
Author(s):  
Bruna Neves ◽  
Pedro Domingues ◽  
Maria Oliveira ◽  
Maria Domingues ◽  
Tânia Melo
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Head Group ◽  
Fatty Acyl Chain ◽  
Polar Head Group ◽  
Derivatives Of

Nitrated lipids have been detected in vitro and in vivo, usually associated with a protective effect. While nitrated fatty acids have been widely studied, few studies reported the nitration and nitroxidation of the phospholipid classes phosphatidylcholine, and phosphatidylethanolamine. However, no information regarding nitrated and nitroxidized phosphatidylserine can be found in the literature. This work aims to identify and characterize the nitrated and nitroxidized derivatives of 1-palmitoyl-2-oleoyl-sn-3-glycero-phosphoserine (POPS), obtained after incubation with nitronium tetrafluoroborate, by liquid chromatography (LC) coupled to mass spectrometry (MS) and tandem MS (MS/MS). Several nitrated and nitroxidized products were identified, namely, nitro, nitroso, nitronitroso, and dinitro derivatives, as well as some nitroxidized species such as nitrosohydroxy, nitrohydroxy, and nitrohydroperoxy. The fragmentation pathways identified were structure-dependent and included the loss of HNO and HNO2 for nitroso and nitro derivatives, respectively. Combined losses of PS polar head group plus HNO or HNO2 and carboxylate anions of modified fatty acyl chain were also observed. The nitrated POPS also showed antiradical potential, demonstrated by the ability to scavenge the ABTS●+ and DPPH● radicals. Overall, this in vitro model of nitration based on LC-MS/MS provided additional insights into the nitrated and nitroxidized derivatives of PS and their fragmentation fingerprinting. This information is a valuable tool for targeted analysis of these modified PS in complex biological samples, to further explore the new clues on the antioxidant potential of nitrated POPS.

scholarly journals Cellular Lipid Composition Affects Sensitivity of Plant Pathogens to Fengycin, an Antifungal Compound Produced by Bacillus subtilis Strain CU12

2014 ◽  
Vol 104 (10) ◽  
pp. 1036-1041 ◽  
Author(s):  
Cody Wise ◽  
Justin Falardeau ◽  
Ingrid Hagberg ◽  
Tyler J. Avis
Keyword(s):  
Bacillus Subtilis ◽  
Lipid Composition ◽  
Plant Pathogens ◽  
Membrane Lipids ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Subtilis Strain ◽  
Antifungal Compound ◽  
Membrane Pore ◽  
Ergosterol Content

Fengycin is an antimicrobial cyclic lipopeptide produced by various Bacillus subtilis strains, including strain CU12. Direct effects of fengycin include membrane pore formation and efflux of cellular contents leading to cell death in sensitive microorganisms. In this study, four plant pathogens were studied in order to elucidate the role of membrane lipids in their relative sensitivity to fengycin. Inhibition of mycelial growth in these pathogens varied considerably. Analysis of membrane lipids in these microorganisms indicated that sensitivity correlated with low ergosterol content and shorter phospholipid fatty acyl chains. Sensitivity to fengycin also correlated with a lower anionic/zwitterionic phospholipid ratio. Our data suggest that decreased fluidity buffering capacity, as a result of low ergosterol content, and higher intrinsic fluidity afforded by short fatty acyl chain length may increase the sensitivity of microbial membranes to fengycin. Our results also suggest that lower content in anionic phospholipids may increase fengycin insertion into the membrane through reduced electrostatic repulsion with the negatively charged fengycin. The intrinsic membrane lipid composition may contribute, in part, to the observed level of antimicrobial activity of fengycin in various plant pathogens.

scholarly journals Ectopic expression of ceramide synthase 2 in neurons suppresses neurodegeneration induced by ceramide synthase 1 deficiency

2016 ◽  
Vol 113 (21) ◽  
pp. 5928-5933 ◽  
Author(s):  
Stefka D. Spassieva ◽  
Xiaojie Ji ◽  
Ye Liu ◽  
Kenneth Gable ◽  
Jacek Bielawski ◽  
...  
Keyword(s):  
Acyl Chain ◽  
Strong Association ◽  
Ectopic Expression ◽  
Fatty Acyl ◽  
Chemical Diversity ◽  
Fatty Acyl Chain ◽  
Length Variation ◽  
Ceramide Synthase ◽  
Hydrophobic Moiety

Sphingolipids exhibit extreme functional and chemical diversity that is in part determined by their hydrophobic moiety, ceramide. In mammals, the fatty acyl chain length variation of ceramides is determined by six (dihydro)ceramide synthase (CerS) isoforms. Previously, we and others showed that mutations in the major neuron-specific CerS1, which synthesizes 18-carbon fatty acyl (C18) ceramide, cause elevation of long-chain base (LCB) substrates and decrease in C18 ceramide and derivatives in the brain, leading to neurodegeneration in mice and myoclonus epilepsy with dementia in humans. Whether LCB elevation or C18 ceramide reduction leads to neurodegeneration is unclear. Here, we ectopically expressed CerS2, a nonneuronal CerS producing C22–C24 ceramides, in neurons of Cers1-deficient mice. Surprisingly, the Cers1 mutant pathology was almost completely suppressed. Because CerS2 cannot replenish C18 ceramide, the rescue is likely a result of LCB reduction. Consistent with this hypothesis, we found that only LCBs, the substrates common for all of the CerS isoforms, but not ceramides and complex sphingolipids, were restored to the wild-type levels in the Cers2-rescued Cers1 mutant mouse brains. Furthermore, LCBs induced neurite fragmentation in cultured neurons at concentrations corresponding to the elevated levels in the CerS1-deficient brain. The strong association of LCB levels with neuronal survival both in vivo and in vitro suggests high-level accumulation of LCBs is a possible underlying cause of the CerS1 deficiency-induced neuronal death.

Optimization of Phosphatidylserine-Modified Titanium Surfaces for Enhanced Osteoblast Response

2006 ◽  
Author(s):  
Neera Satsangi ◽  
Arpan Satsangi ◽  
Joo L. Ong ◽  
Rajiv V. Satsangi
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Production ◽  
Specific Activity ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Separate Experiment ◽  
Calcium Deposition ◽  
Fatty Acyl Chain ◽  
Acyl Chain Length

This report is part of a continued effort to evaluate the in vitro osteoblast responses on different phospholipid coatings on Titanium (Ti) implant materials. It has been established that, among analogous phopholipids, the Ti surfaces coated with calcium phosphate (CaP) complex of phosphatidylserine induce the best calcium deposition and osteoblast growth and metabolism. This communication describes an effort to optimize the chemical structure of phosphatidylserine at its position−1 and −2, as Ti surface coating relative to enhancement in osteoblast differentiation and growth in culture. Four synthetic phosphatidylserine analogs with varying fatty acyl chain length and unsaturation were converted to CaP complex, coated on Ti discs, and the osteoblast progenitor cells were cultured on them for up to 14 days to study their differentiation, growth and biochemistry as marked by the expression of alkaline phosphatase specific activity and protein production. In a separate experiment, the topography of the glass surface (glass Petri-dishes) coated the analogous phosphatidylserines, after immersion in simulated body fluid, was examined by scanning electron microscopy (SEM). The presence of calcium and phosphate ions in this deposit was also confirmed. The inclusion of unsaturation in fatty acyl chain in phosphatidylserine enhanced the Total protein production (TPP) as well as the alkaline phosphatase (ALP) specific activity.

scholarly journals Fatty Acyl Benzamido Antibacterials Based on Inhibition of DnaK-catalyzed Protein Folding

2006 ◽  
Vol 282 (7) ◽  
pp. 4437-4446 ◽  
Author(s):  
Markus Liebscher ◽  
Günther Jahreis ◽  
Christian Lücke ◽  
Susanne Grabley ◽  
Satish Raina ◽  
...  
Keyword(s):  
Protein Folding ◽  
Minimal Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Secondary Amide ◽  
Fatty Acyl Chain ◽  
Isomerase Activity

We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. Nα-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 ± 20 μg/ml. The compounds were shown to compete with fluorophore-labeled σ32-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.

scholarly journals Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement

2016 ◽  
Vol 27 (16) ◽  
pp. 2590-2597 ◽  
Author(s):  
Michael Zick ◽  
William Wickner
Keyword(s):  
Acyl Chain ◽  
Fatty Acyl ◽  
Fatty Acyl Chain ◽  
Rab Gtpase ◽  
Lower Phase ◽  
Yeast Vacuole ◽  
Vacuole Fusion ◽  
In Vitro Reconstitution

In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1.

scholarly journals Biophysical characterization and crystallization of the membrane protein Etk

2014 ◽  
Vol 70 (a1) ◽  
pp. C1499-C1499
Author(s):  
Chelsy Prince ◽  
Zongchao Jia
Keyword(s):  
Membrane Protein ◽  
Capsular Polysaccharide ◽  
Analytical Ultracentrifugation ◽  
Colorimetric Assay ◽  
Kinase Domain ◽  
Full Length ◽  
Size Exclusion ◽  
Head Group ◽  
Minimal Sample

The inner membrane protein E. coli tyrosine kinase (Etk) is part of a large protein complex that assembles and exports capsular polysaccharide (CPS) in Gram-negative bacteria. Etk interacts with an outer membrane protein channel, YccZ (1), and regulates CPS export through autophosphorylation of a tyrosine cluster in its C-terminal tail. Previous work resulted in the structure of the isolated Etk C-terminal kinase domain (2). In the present study, the full-length protein has been purified and characterized in vitro. When purified in n-dodecyl-β-D-maltoside (DDM), Etk full length retains autophosphorylation activity, but is not suitable for crystallization because it severely aggregates and degrades. Using the main degradation product, a truncation containing the N-terminal domain (interacts with YccZ) and both transmembrane helices was designed. Truncated Etk does not further degrade and exists as a mixture of monomers and dimers when solubilized by five detergents as determined by size-exclusion chromatography and analytical ultracentrifugation. Crystals have been successfully grown when the protein is solubilized in DDM or n-decyl-β-D-maltoside (DM). The most promising crystals (DDM, 0.1 M MES pH 6.0, 1-5% PEG 3000, 20-30% PEG 200) have been reproduced and optimized with the assistance of a colorimetric assay (3). This assay relies on a reaction between 2,6-dimethylphenol, sulfuric acid, and the sugar head group of certain detergents to accurately quantify detergent in crystallization samples with minimal sample loss. Additive screening also revealed that MgCl2 improves crystallization. Optimization of this crystallization condition has significantly improved reproducibility of these crystals, but x-ray diffraction is limited to 6.5 Å. Current work is focused on reproducing and optimizing a second crystallization lead (DM, 0.1 M KH2PO4 pH 7.5, 32% PEG 400, 0.1 M KCl).

scholarly journals Regulatory effects of fatty acyl-coenzyme A derivatives on phosphate-activated pig brain and kidney glutaminase in vitro

1975 ◽  
Vol 149 (1) ◽  
pp. 83-91 ◽  
Author(s):  
E Kvamme ◽  
I A Torgner
Keyword(s):  
Acyl Chain ◽  
Bromothymol Blue ◽  
Fatty Acyl ◽  
Fatty Acyl Chain ◽  
Pig Brain ◽  
Low Concentrations ◽  
Regulatory Effects ◽  
Acyl Coenzyme A ◽  
Carnitine Derivatives

1. Fatty n-acyl-CoA derivatives in the concentration range 5 μM-0.1mM and with 5-18 fatty acyl carbons have dual effects on phosphate-activated glutaminase from pig brain and kidney. Generally, fatty acyl-CoA derivatives in low concentrations activate the enzyme, but inhibit at higher concentrations; phosphate and citrate potentiate the activation, displaying positive co-operatively, and protect against inactivation. The fatty acyl-CoA derivatives affect glutaminase similarly to Bromothymol Blue, but differently from acetyl-CoA, which activates the enzyme only at very low phosphate or citrate concentrations. 2. Saturated fatty acyl-CoA derivatives, with 5-10 fatty acyl carbons, only activate the enzyme in the concentration range 0-0.1 mM. When the fatty acyl chain is elongated, the fatty acyl-CoA derivatives gradually become more powerful inhibitors of glutaminase at the expense of their activating capacity. In particular, palmitoyl-CoA and stearoyl-CoA are strong inhibitors at concentrations (10 μM) at which the corresponding free fatty acids and fatty acyl-carnitine derivatives have no effect. 3. The unsaturated fatty acyl-CoA derivatives, oleoyl-CoA and linoleoyl-CoA, behave as potent activators in the lower part of the concentration range tested (0-0.05mM), and as inhibitors in the upper part of this range (0.02-0.10mM). Oleic acid and linoleic acid have similar properties, but their activating capacity is less pronounced. 4. Phosphate both prevented and reversed the inhibition, but no restoration of activity was possible once the enzyme became inactivated. 5. By changing the pH from 7.0 to 8.0 the activating capacity of the fatty acyl-CoA derivatives is increased, as is their concentration range for activation. 6. The fatty acyl-CoA derivatives are somewhat more potent activator for brain glutaminase, but otherwise they affect the two enzymes similarly.

Uncoupling of cardiac cells by doxyl stearic acids specificity and mechanism of action

1989 ◽  
Vol 256 (4) ◽  
pp. C913-C924 ◽  
Author(s):  
J. M. Burt
Keyword(s):  
Single Channel ◽  
Methyl Esters ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Head Group ◽  
Fatty Acyl Chain ◽  
Heart Cells ◽  
Dependent Manner

The influence of doxyl stearic acids (DSAs) on gap junctional conductance (gj) between pairs of neonatal rat heart cells was studied. DSAs are spin probes that perturb the membrane at different depths depending on position of the doxyl group on the fatty acyl chain. 16-DSA and 12-DSA rapidly and reversibly reduced gj to unmeasureable levels in a dose- and time-dependent manner. Single channel events observed when gj was low were of the same unitary size as those observed under control conditions. The methyl esters of 16- and 12-DSA, stearic acid itself, and TEMPO, an analogue of the doxyl group that has no fatty acyl chain, had no effect on gj. Protonation of the carboxyl head group (by acidifying the solution) reduced the potency of 16- or 12-DSA. Spontaneous beating activity and action potentials were observed at concentrations of the DSAs 15-20 times that necessary for uncoupling. These results indicate that uncoupling by the DSAs requires the presence of the charged carboxyl group and localized perturbation of the channel at the lipid-channel interface by the doxyl group. Furthermore, they predict that unsaturated free fatty acids, which accumulate during ischemia, may exert their arrhythmogenic effect by reducing gj, and thereby slowing conduction.

Intestinal microsomes: polyunsaturated fatty acid metabolism and regulation of enterocyte transport properties

1990 ◽  
Vol 68 (5) ◽  
pp. 636-641 ◽  
Author(s):  
M. L. Garg ◽  
M. Keelan ◽  
A. B. R. Thomson ◽  
M. T. Clandinin
Keyword(s):  
Acyl Chain ◽  
Lipid Synthesis ◽  
Fatty Acyl ◽  
Fine Tuning ◽  
Membrane Properties ◽  
Head Group ◽  
Fatty Acyl Chain ◽  
Polar Head Group ◽  
Intestinal Microsomes ◽  
Main Determinants

Recent evidence has suggested that transport of nutrients from the lumen to the interior of the gastrointestinal epithelium and exit of nutrients from the enterocyte to the circulation is governed by physicochemical properties of brush border and basolateral membranes, respectively. The main determinants of membrane properties are phospholipid, cholesterol, and fatty acyl chain composition (chain length and degree of unsaturation). Lipid synthesis occurs in enterocyte microsomes and the fine tuning of lipid composition is done at other subcellular sites by deacylation–reacylation or by changing the polar head group (e.g., by phosphatidylethanolamine methyltransferase). The present paper will focus on the mechanisms by which enterocyte membranes adapt functional properties in response to external stimuli. It is proposed that under the influence of internal or external stress, the enzymes of lipid metabolism in microsomes are modulated. These changes in lipid synthesis are reflected in other subcellular membranes, changing their physicochemical status and thus transport phenomena. One of the initial events appears to be alteration in desaturase enzyme activity. Our results suggest that desaturase activity and the fatty acyl profiles of the intestinal mucosal phospholipid rapidly respond to physiological conditions such as fasting and dietary fat treatment.Key words: polyunsaturated fatty acids, desaturases, enterocyte, intestinal microsomes, adaptation.

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