scholarly journals Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

2018 ◽  
Author(s):  
M. Doktorova ◽  
F. A. Heberle ◽  
D. Marquardt ◽  
R. Rusinova ◽  
L. Sanford ◽  
...  
Keyword(s):  
Effective Means ◽  
Transmembrane Proteins ◽  
Lipid Vesicles ◽  
Melting Transition ◽  
Dependent Manner ◽  
Scanning Calorimetry ◽  
Concentration Dependent Manner ◽  
Flip Flop ◽  
Lipid Distribution ◽  
Dynamics Simulations

ABSTRACTUnlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Non-enzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels which have well-defined structures and known function, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small-angle X-ray scattering, circular dichroism and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time suggestive of scrambling activity, and the effect of the channel on the transbilayer lipid distribution in both symmetric POPC and asymmetric POPC/DMPC vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the amount of membrane deformation in the samples. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

scholarly journals Size of Cells and Physicochemical Properties of Membranes are Related to Flavor Production during Sake Brewing in the Yeast Saccharomyces cerevisiae

Membranes ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 440
Author(s):  
Tsuyoshi Yoda ◽  
Tomoaki Saito
Keyword(s):  
Isoamyl Alcohol ◽  
Lipid Vesicles ◽  
Caproic Acid ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Concentration Dependent Manner ◽  
Generalized Polarization ◽  
Flavor Components ◽  
The Impact

Ethyl caproate (EC) and isoamyl acetate (IA) are key flavor components of sake. Recently, attempts have been made to increase the content of good flavor components, such as EC and IA, in sake brewing. However, the functions of EC and IA in yeast cells remain poorly understood. Therefore, we investigated the effects of EC and IA using cell-sized lipid vesicles. We also investigated lipid vesicles containing EC and/or caproic acid (CA) as well as IA and/or isoamyl alcohol (IAA). CA and IAA are precursors of EC and IA, respectively, and are important flavors in sake brewing. The size of a vesicle is influenced by flavor compounds and their precursors in a concentration-dependent manner. We aimed to establish the conditions in which the vesicles contained more flavors simultaneously and with different ratios. Interestingly, vesicles were largest in a mixture of 50% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with 25% EC and 25% CA or a mixture of 50% DOPC with 25% IA and 25% IAA. The impact of flavor additives on membrane fluidity was also studied using Laurdan generalized polarization. During the production process, flavors may regulate the fluidity of lipid membranes.

TPCK Induces Apoptosis in Human Acute Myeloid Leukemia U-937 Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4478-4478
Author(s):  
Julia Mazar ◽  
Alexandra Lichtenstein ◽  
Leora Katz ◽  
Ofer Shpilberg ◽  
Itai Levi ◽  
...  
Keyword(s):  
Chromatin Condensation ◽  
Annexin V ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Concentration Dependent Manner ◽  
Flip Flop ◽  
Induced Apoptosis

Abstract Many types of antitumor therapy in general and AML in particular exert their effect by activating apoptosis. Apoptosis of AML cells can be induced by cytostatic drugs, corticosteroids, and radiation. Recently, the role of different proteases as possible targets for chemotherapy was described. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a chymotrypsin-like protease (CLP) inhibitor was shown to exert a dual effect on leukemic cells: proapoptotic and antiapoptotic. In the present study the mechanism of its proapoptotic effect was addressed. It was found that the CLP inhibitors, TPCK and 3,4 dichloroisicoumarine induced apoptosis in a time- and concentration-dependent manner. Apoptosis was accompanied by a decrease in mitochondrial membrane potential, cytochrome c release, caspase-3 (but not caspase-8) activation, PS flip-flop (measured by Annexin-V staining followed by flow cytometry analysis) and chromatin condensation, but not fragmentation (detected by acridine orange/ethidium bromide staining). All apoptotic processes induced by TPCK were completely inhibited by cycloheximide. The ability of cycloheximide to inhibit TPCK-induced cell death suggests that protein synthesis plays a role in TPCK-induced apoptosis. Additionaly, the proapoptotic effect of TPCK was abolished by elimination of glucose from the medium. The data supports the role of mitochondria in this process. In the present study the apoptotic pathway driven by inhibition of CLP was demonstrated. Moreover, these findings suggest possible ways of preventing the proapoptotic activity of TPCK and thereby enhancimg its antiapoptotic action.

scholarly journals Influence of Ethyl Caproate on the Size of Lipid Vesicles and Yeast Cells

Biomimetics ◽  
2020 ◽  
Vol 5 (2) ◽  
pp. 16 ◽  
Author(s):  
Tsuyoshi Yoda ◽  
Akira Ogura ◽  
Tomoaki Saito
Keyword(s):  
Stationary Phase ◽  
Cell Size ◽  
Lipid Vesicles ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Vesicle Size ◽  
Flavor Component ◽  
Aromatic Components ◽  
Ethyl Caproate

Ethyl caproate (EC) is a key flavor component of sake. Recently, in sake brewing, an effort has been underway to increase the content of aromatic components such as EC. However, the function of EC in yeast cells remains poorly understood. Therefore, we investigated the effects of EC using cell-sized lipid vesicles. We found that vesicle size decreases in a concentration-dependent manner when EC is contained in lipid vesicles. Furthermore, yeast experiments showed that a strain producing high quantities of EC in its stationary phase decreased in size during EC production. Given caproic acid’s (CA) status as the esterification precursor of EC in yeast, we also compared lipid vesicles containing CA with those containing EC. We found that CA vesicles were smaller than EC vesicles of the same concentration. These results suggest that EC production may function apparently to maintain cell size.

scholarly journals Molecular mechanism for NLRP6 inflammasome assembly and activation

2019 ◽  
Vol 116 (6) ◽  
pp. 2052-2057 ◽  
Author(s):  
Chen Shen ◽  
Alvin Lu ◽  
Wen Jun Xie ◽  
Jianbin Ruan ◽  
Roberto Negro ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Conformational Changes ◽  
Protein Complexes ◽  
Multiple Roles ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pyrin Domain ◽  
Dynamics Simulations ◽  
Inflammatory Caspases ◽  
Lrr Domain

Inflammasomes are large protein complexes that trigger host defense in cells by activating inflammatory caspases for cytokine maturation and pyroptosis. NLRP6 is a sensor protein in the nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing (NLR) inflammasome family that has been shown to play multiple roles in regulating inflammation and host defenses. Despite the significance of the NLRP6 inflammasome, little is known about the molecular mechanism behind its assembly and activation. Here we present cryo-EM and crystal structures of NLRP6 pyrin domain (PYD). We show that NLRP6 PYD alone is able to self-assemble into filamentous structures accompanied by large conformational changes and can recruit the ASC adaptor using PYD–PYD interactions. Using molecular dynamics simulations, we identify the surface that the NLRP6 PYD filament uses to recruit ASC PYD. We further find that full-length NLRP6 assembles in a concentration-dependent manner into wider filaments with a PYD core surrounded by the NBD and the LRR domain. These findings provide a structural understanding of inflammasome assembly by NLRP6 and other members of the NLR family.

scholarly journals Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes

2020 ◽  
Vol 6 (8) ◽  
pp. eaay5736 ◽  
Author(s):  
Eiji Yamamoto ◽  
Jan Domański ◽  
Fiona B. Naughton ◽  
Robert B. Best ◽  
Antreas C. Kalli ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Bound States ◽  
Lipid Binding ◽  
Dependent Manner ◽  
Ph Domain ◽  
Concentration Dependent Manner ◽  
Phosphatidylinositol Phosphate ◽  
Ph Domains ◽  
Experimental Values ◽  
Dynamics Simulations

Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

1993 ◽  
Vol 69 (03) ◽  
pp. 286-292 ◽  
Author(s):  
Che-Ming Teng ◽  
Feng-Nien Ko ◽  
Inn-Ho Tsai ◽  
Man-Ling Hung ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Snake Venom ◽  
Inositol Phosphate ◽  
Shape Change ◽  
Platelet Activating Factor ◽  
Atp Release ◽  
Platelet Membrane ◽  
Thromboxane B2 ◽  
Dependent Manner ◽  
Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

Anticoagulant Activity of β2-Glycoprotein I Is Potentiated by a Distinct Subgroup of Anticardiolipin Antibodies

1992 ◽  
Vol 68 (03) ◽  
pp. 297-300 ◽  
Author(s):  
Monica Galli ◽  
Paul Comfurius ◽  
Tiziano Barbui ◽  
Robert F A Zwaal ◽  
Edouard M Bevers
Keyword(s):  
Human Plasma ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Type A ◽  
Lipid Vesicles ◽  
Dependent Manner ◽  
Type B ◽  
Distinct Subgroup ◽  
Glycoprotein I ◽  
Thrombin Formation

SummaryPlasmas of 16 patients positive for both IgG anticardiolipin (aCL) antibodies and lupus anticoagulant (LA) antibodies were subjected to adsorption with liposomes containing cardiolipin. In 5 of these plasmas both the anticardiolipin and the anticoagulant activities were co-sedimented with the liposomes in a dose-dependent manner, whereas in the remaining cases only the anticardiolipin activity could be removed by the liposomes, leaving the anticoagulant activity (LA) in the supernatant plasma. aCL antibodies purified from the first 5 plasmas were defined as aCL-type A, while the term aCL-type B was used for antibodies in the other 11 plasmas, from which 2 were selected for this study.Prolongation of the dRVVT was produced by affinity-purified aCL-type A antibodies in plasma of human as well as animal (bovine, rat and goat) origin. aCL-type B antibodies were found to be devoid of anticoagulant activity, while the corresponding supernatants containing LA IgG produced prolongation of the dRVVT only in human plasma.These anticoagulant activities of aCL-type A and of LA IgG's were subsequently evaluated in human plasma depleted of β2-glycoprotein I (β2-GPI), a protein which was previously shown to be essential in the binding of aCL antibodies to anionic phospholipids. Prolongation of the dRVVT by aCL-type A antibodies was abolished using β2-GPI deficient plasma, but could be restored upon addition of β2-GPI. In contrast, LA IgG caused prolongation of the dRVVT irrespective of the presence or absence of β2-GPI.Since β2-GPI binds to negatively-charged phospholipids and impedes the conversion of prothrombin by the factor Xa/Va enzyme complex (Nimpf et al., Biochim Biophys Acta 1986; 884: 142–9), comparison was made of the effect of aCL-type A and aCL-type B antibodies on the rate of thrombin formation in the presence and absence of β2-GPI. This was measured in a system containing highly purified coagulation factors Xa, Va and prothrombin and lipid vesicles composed of 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. No inhibition on the rate of thrombin formation was observed with both types of aCL antibodies when either β2-GPI or the lipid vesicles were omitted. Addition of β2-GPI to the prothrombinase assay in the presence of lipid vesicles causes a time-dependent inhibition which was not affected by the presence of aCL-type B or non-specific IgG. In contrast, the presence of aCL-type A antibodies dramatically increased the anticoagulant effect of β2-GPI. These data indicate that the anticoagulant activity of aCL-type A antibodies in plasma is mediated by β2-GPI.

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