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1.
Biochemistry (Mosc) ; 84(2): 164-170, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31216975

RESUMO

Quantitative and qualitative assessments of cell membrane components are essential for the accurate interpretation of processes occurring in biological membranes. Changes in the structure and function of cell membrane components have been linked to oxidative stress. Oxidative stress induced by chronic ethanol consumption or cancer transformation has been implicated in changing the levels of phospholipids and fatty acids in the cell membrane. In this study, we used high-performance liquid chromatography to quantitate the effects of alcohol and malignant transformation on membrane components, namely phospholipids and free fatty acids. Ethanol increased the phospholipid levels. Moreover, the process of malignant transformation was accompanied by increased levels of phospholipids and arachidonic acid as well as decreased levels of linoleic acid and α-linolenic acid. Thus, these oxidative stress-inducing conditions that cause variations in the cellular composition affect the actions of the cell membrane and cell function.


Assuntos
Membrana Celular/metabolismo , Fosfolipídeos/metabolismo , Administração Oral , Intoxicação Alcoólica/tratamento farmacológico , Animais , Ácido Araquidônico/química , Ácido Araquidônico/isolamento & purificação , Ácido Araquidônico/metabolismo , Etanol/administração & dosagem , Etanol/toxicidade , Masculino , Estresse Oxidativo , Fosfolipídeos/química , Fosfolipídeos/isolamento & purificação , Ratos , Ratos Wistar
2.
Environ Sci Pollut Res Int ; 26(22): 22197-22208, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31148000

RESUMO

Acrylamide (ACR), a ubiquitous agent, has various chemical and industrial applications, and it is found in backed or fried carbohydrate-rich food. It has been related to multiple toxicological effects, and it causes high cytotoxicity through oxidative stress. The present study aimed to investigate the potential effect of ACR toxicity administered at different concentrations (5, 10, and 20 mg/L), during 5 days, in order to evaluate the fatty acid (FA) composition and redox state in the digestive gland of Mactra corallina. The results showed, in ACR-treated clams, a significant increase in malondialdehyde, hydrogen peroxide, protein carbonyl, and metallothionein levels, as well as an alteration of the enzymatic (superoxide dismutase, glutathione peroxidase, and catalase) and non-enzymatic (reduced glutathione and ascorbic acid) antioxidant status. However, acetylcholinesterase activity was inhibited in a concentration-dependent manner. In our experiment, the n-3 (Omega-3) and n-6 (Omega-6) polyunsaturated fatty acid levels were significantly changed in all ACR-treated groups. A decrease in eicosapentaenoic acid (C20:5n-3, EPA) and docosahexaenoic acid (C22:6n-3, DHA) was observed in 10-mg/L and 20-mg/L ACR-treated groups. Nevertheless, arachidonic acid (C20:4n-6, ARA) and its precursor linoleic acid (C18:2n-6, LA) were increased. Besides oxidative stress parameters, FA composition may be an additional tool for assessing ACR contamination.


Assuntos
Acrilamida/farmacologia , Bivalves/metabolismo , Digestão/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos/metabolismo , Superóxido Dismutase/metabolismo , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Ácido Araquidônico/química , Bivalves/química , Catalase/metabolismo , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/química , Glutationa/metabolismo , Ácido Linoleico/química , Ácido Linoleico/farmacologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos
3.
Methods Mol Biol ; 1982: 75-101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172467

RESUMO

The NADPH oxidase NOX2 complex consists of assembled cytosolic and redox membrane proteins. In mammalian cells, natural arachidonic acid (cis-AA), released by activated phospholipase-A2, plays an important role in the activation of the NADPH oxidase, but the mechanism of action of cis-AA is still a matter of debate. In cell-free systems, cis-AA is commonly used for activation although its structural effects are still unclear. Undoubtedly cis-AA participates in the synergistic multi-partner assembly that can be hardly studied at the molecular level in vivo due to cellular complexity. The capacity of this anionic amphiphilic fatty acid to activate the oxidase is mainly explained by its ability to disrupt intramolecular bonds, mimicking phosphorylation events in cell signaling and therefore allowing protein-protein interactions. Interestingly the geometric isomerism of the fatty acid and its purity are crucial for optimal superoxide production in cell-free assays. Indeed, optimal NADPH oxidase assembly was hampered by the substitution of the cis form by the trans forms of AA isomers (Souabni et al., BBA-Biomembranes 1818:2314-2324, 2012). Structural analysis of the changes induced by these two compounds, by circular dichroism and by biochemical methods, revealed differences in the interaction between subunits. We describe how the specific geometry of AA plays an important role in the activation of the NOX2 complex.


Assuntos
Ácido Araquidônico/metabolismo , NADPH Oxidases/metabolismo , Fagócitos/enzimologia , Ácido Araquidônico/química , Fracionamento Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Sistema Livre de Células , Colorimetria , Ativação Enzimática , Isomerismo , Estrutura Molecular , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/química , NADPH Oxidases/isolamento & purificação , Neutrófilos/enzimologia , Fagócitos/imunologia , Proteínas Recombinantes de Fusão , Análise Espectral
4.
Int J Mol Sci ; 20(7)2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30935072

RESUMO

Although various ω-3 fatty acid desaturases (ω3Des) have been identified and well-studied regarding substrate preference and regiospecificity, the molecular mechanism of their substrate specificities remains to be investigated. Here we compared two ω3Des, FADS15 from Mortierella alpina and oRiFADS17 from Rhizophagus irregularis, which possessed a substrate preference for linoleic acid and arachidonic acid, respectively. Their sequences were divided into six sections and a domain-swapping strategy was used to test the role of each section in catalytic activity. Heterologous expression and fatty acid experiments of hybrid enzymes in Saccharomyces cerevisiae INVSc1 indicated that the sequences between his-boxes I and II played critical roles in influencing substrate preference. Based on site-directed mutagenesis and molecular docking, the amino acid substitutions W129T and T144W, located in the upper part of the hydrocarbon chain, were found to be involved in substrate specificity, while V137T and V152T were confirmed to interfere with substrate recognition. This study provides significant insight into the structure-function relationship of ω3Des.


Assuntos
Ácidos Graxos Dessaturases/química , Proteínas Fúngicas/química , Glomeromycota/enzimologia , Simulação de Acoplamento Molecular , Mortierella/enzimologia , Ácido Araquidônico/química , Sítios de Ligação , Ácidos Graxos Dessaturases/metabolismo , Proteínas Fúngicas/metabolismo , Ácido Linoleico/metabolismo , Ligação Proteica , Especificidade por Substrato
5.
Food Chem ; 283: 331-337, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30722880

RESUMO

Structured TAGs with palmitic acid and polyunsaturated fatty acid (PUFA) at the sn-2 position have various health benefits for infants. In this study, we first compared two enzymatic routes for preparation of the structured TAGs. Results showed that the one-pot and two-step syntheses led to 37.6% and 55.4% oleic acid incorporation, respectively, after 10 h and reaction route had little effect on the sn-2 fatty acid composition. Subsequently, reaction variables of the two-step synthesis were optimized. Under the optimal conditions, 53.5% oleic acid was incorporated into the structured TAGs after 6-h acidolysis. Major fatty acids at the sn-2 position were palmitic acid (68.7%), ARA (9.8%) and oleic acid (7.9%). This is the first study reporting a two-step enzymatic method for structured TAGs preparation. Compared to the one-pot synthesis, current method significantly improves the efficiency of the acidolysis by product inhibition elimination. The synthetic TAGs have potential use in infant formulas.


Assuntos
Ácido Araquidônico/metabolismo , Fórmulas Infantis/análise , Ácidos Palmíticos/metabolismo , Triglicerídeos/química , Ácido Araquidônico/química , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Humanos , Lactente , Lipase/metabolismo , Ácido Oleico/química , Ácido Oleico/metabolismo , Ácidos Palmíticos/química , Triglicerídeos/metabolismo
6.
Thromb Haemost ; 119(4): 567-575, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30769363

RESUMO

Endocannabinoids are a group of arachidonic acid-derived lipid mediators binding to cannabinoid receptors CB1 and CB2. An overactivity of the endocannabinoid system plays a pathophysiological role in the development of visceral obesity and insulin resistance. Moreover, elevated circulating endocannabinoid levels are also prevalent in atherosclerosis. The pathophysiological increase of endocannabinoid levels is due to an altered expression of endocannabinoid synthesizing and degrading enzymes induced by inflammatory mediators such as cytokines or lipids. Emerging experimental evidence suggests that enhanced endocannabinoid signalling affects atherosclerosis via multiple effects, including a modulation of vascular inflammation, leukocyte recruitment, macrophage cholesterol metabolism and consequently atherosclerotic plaque stability. In addition, recent findings in various metabolic disease models highlight the relevance of peripheral CB1 cannabinoid receptors in adipose tissue, liver and pancreas, which crucially regulate lipid and glucose metabolism as well as macrophage properties in these organs. This suggests that targeting the endocannabinoid system in the vasculature and peripheral organs might have a therapeutic potential for atherosclerosis by inhibiting vascular inflammation and improving metabolic risk factors. This review will provide a brief update on the effects of endocannabinoid signalling in atherosclerosis and related metabolic complications.


Assuntos
Aterosclerose/metabolismo , Endocanabinoides/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Tecido Adiposo/metabolismo , Animais , Ácido Araquidônico/química , Ácidos e Sais Biliares/química , Glicemia/metabolismo , Sistema Cardiovascular/metabolismo , Citocinas/metabolismo , Humanos , Inflamação , Resistência à Insulina , Ligantes , Metabolismo dos Lipídeos , Lipídeos/química , Fígado/metabolismo , Camundongos , Camundongos Knockout , Obesidade Abdominal/metabolismo , Pâncreas/metabolismo , Receptores de Canabinoides/metabolismo , Fatores de Risco
7.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670555

RESUMO

Talaromyces marneffei infection causes talaromycosis (previously known as penicilliosis), a very important opportunistic systematic mycosis in immunocompromised patients. Different virulence mechanisms in T. marneffei have been proposed and investigated. In the sera of patients with talaromycosis, Mp1 protein (Mp1p), a secretory galactomannoprotein antigen with two tandem ligand-binding domains (Mp1p-LBD1 and Mp1p-LBD2), was found to be abundant. Mp1p-LBD2 was reported to possess a hydrophobic cavity to bind copurified palmitic acid (PLM). It was hypothesized that capturing of lipids from human hosts by expressing a large quantity of Mp1p is a virulence mechanism of T. marneffei It was shown that expression of Mp1p enhanced the intracellular survival of T. marneffei by suppressing proinflammatory responses. Mechanistic study of Mp1p-LBD2 suggested that arachidonic acid (AA), a precursor of paracrine signaling molecules for regulation of inflammatory responses, is the major physiological target of Mp1p-LBD2. In this study, we use crystallographic and biochemical techniques to further demonstrate that Mp1p-LBD1, the previously unsolved first lipid binding domain of Mp1p, is also a strong AA-binding domain in Mp1p. These studies on Mp1p-LBD1 support the idea that the highly expressed Mp1p is an effective AA-capturing protein. Each Mp1p can bind up to 4 AA molecules. The crystal structure of Mp1p-LBD1-LBD2 has also been solved, showing that both LBDs are likely to function independently with a flexible linker between them. T. marneffei and potentially other pathogens highly expressing and secreting proteins similar to Mp1p can severely disturb host signaling cascades during proinflammatory responses by reducing the availabilities of important paracrine signaling molecules.


Assuntos
Ácido Araquidônico/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Micoses/microbiologia , Talaromyces/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Ácido Araquidônico/química , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno , Humanos , Espectrometria de Massas , Micoses/genética , Micoses/imunologia , Domínios Proteicos , Talaromyces/química , Talaromyces/genética , Fatores de Virulência/genética
8.
Cell Chem Biol ; 26(3): 420-432.e9, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30686757

RESUMO

The initiation and execution of cell death can be regulated by various lipids. How the levels of environmental (exogenous) lipids impact cell death sensitivity is not well understood. We find that exogenous monounsaturated fatty acids (MUFAs) potently inhibit the non-apoptotic, iron-dependent, oxidative cell death process of ferroptosis. This protective effect is associated with the suppression of lipid reactive oxygen species (ROS) accumulation at the plasma membrane and decreased levels of phospholipids containing oxidizable polyunsaturated fatty acids. Treatment with exogenous MUFAs reduces the sensitivity of plasma membrane lipids to oxidation over several hours. This effect requires MUFA activation by acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) and is independent of lipid droplet formation. Exogenous MUFAs also protect cells from apoptotic lipotoxicity caused by the accumulation of saturated fatty acids, but in an ACSL3-independent manner. Our work demonstrates that ACSL3-dependent MUFA activation promotes a ferroptosis-resistant cell state.


Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Lipídeos/química , Animais , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Coenzima A Ligases/metabolismo , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Camundongos , Oxirredução , /metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo
9.
Microb Pathog ; 126: 56-62, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30393116

RESUMO

This study aimed to investigate the total phenolic content (TPC), the identification of the common compounds by HPLC-ESI-MS and HPLC-ESI-MS-TOF and the inhibitory effects against class A-type ß-lactamase (GES-22 variant, produced recombinantly) in methanolic extracts (MEs) of four Algerian seaweeds [Ulva intestinalis, Codium tomentosum, Dictyota dichotoma and Halopteris scoparia]. The TPC varied among the four species, ranging between 0.93 ±â€¯0.65 and 2.66 ±â€¯1.33 mg GAEs/g DW. C.tomentosum had higher total phenol content than other seaweeds while, all of them inhibited uncompetitively GES-22 activity in a dose-dependent manner. Nitrocefin was used as chromogenic substrate to evaluate the inhibitory effect on GES-22. The methanolic extract of D.dichotoma exhibited significant inhibitory effect on GES-22 (IC50 = 13.01 ±â€¯0.046 µg/mL) more than clavulanate, sulbactam and tazobactam (classical ß-lactam inhibitors) (IC50 = 68.38 ±â€¯0.17 µg/mL, 52.68 ±â€¯0.64 µg/mL, and 29.94 ±â€¯0.01 µg/mL, respectively). IC50 of the other ME of U.intestinalis, C.tomentosum, and H.scoparia were 16.87 ±â€¯0.10 µg/mL, 16.54 ±â€¯0.048 µg/mL, and 25.72 ±â€¯0.15 µg/mL, respectively. Except H. scoparia, other three seaweed extracts showed almost two times or more inhibition on GES-22. Furthermore, four common compounds in these MEs were identified, α-linolenic acid (C18:3ω3), linoleic acid (C18:2ω6), oleic acid (C18:1ω9), the eicosanoid precursors ''arachidonic acid'' (C20:4ω6). Baicalein (C15H10O5) was identified in U.intestinalis and D.dichotoma seaweeds. The fact that all seaweed extracts inhibited the GES-22 better than commercial samples makes these seaweeds candidate for discovering new inhibitors against ß-lactamases. Besides that, they contain important components with potential health benefits.


Assuntos
Extratos Vegetais/antagonistas & inibidores , Alga Marinha/química , beta-Lactamases/efeitos dos fármacos , Argélia , Ácido Araquidônico/química , Clorófitas/química , Ensaios Enzimáticos , Flavanonas/química , Mar Mediterrâneo , Metanol , Ácido Oleico/química , Feófitas/química , Fenóis/química , Ácido alfa-Linoleico/química
10.
Org Biomol Chem ; 16(48): 9319-9333, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30511071

RESUMO

Stereoselective synthesis of Z-configured double bonds is central in organic synthesis due to the presence of such motifs in polyunsaturated fatty acids and many natural products. Traditionally, reductions of internal alkynes or Wittig, Ando or Still-Gennari reactions, are often used for preparing such compounds. The substrate scope is limited for both the Ando and the Still-Gennari reactions, while the Wittig reaction often gives low Z-selectivity for the synthesis of polyunsaturated Z-configured methylene interrupted (skipped) double bonds. Reductions of internal alkynes are challenging due to diminished Z-selectivity, poor catalyst reproducibility and over-reductions. An alternative and highly attractive approach is to employ naturally occurring and commercially available polyunsaturated fatty acids as starting materials. The main advantage of this strategy is the conservation of the multiple Z-configured double bonds present in the starting material, allowing a precise incorporation of the desired double bonds into the final product. In particular, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid have been used for the stereoselective synthesis of polyunsaturated fatty acids, their derivatives and other polyunsaturated natural products. Herein, such efforts are reviewed.


Assuntos
Produtos Biológicos/síntese química , Técnicas de Química Sintética/métodos , Ácidos Graxos Insaturados/síntese química , Alquinos/síntese química , Alquinos/química , Ácido Araquidônico/síntese química , Ácido Araquidônico/química , Produtos Biológicos/química , Ácidos Docosa-Hexaenoicos/síntese química , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/síntese química , Ácido Eicosapentaenoico/química , Ácidos Graxos Insaturados/química , Estereoisomerismo
11.
Biochem Biophys Res Commun ; 505(1): 87-92, 2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30241945

RESUMO

The mammalian paraoxonases (PONs 1, 2 and 3) are a family of esterases that are highly conserved within and between species. They exhibit antioxidant and anti-inflammatory activities. However, their physiological function(s) and native substrates are uncertain. Previous structure-activity relationship studies demonstrate that PONs have a high specificity for lipophilic lactones, suggesting that such compounds may be representative of native substrates. This report describes the ability of PONs to hydrolyze two bioactive δ-lactones derived from arachidonic acid, 5,6-dihydroxy-eicosatrienoic acid lactone (5,6-DHTL) and cyclo-epoxycyclopentenone (cyclo-EC). Both lactones were very efficiently hydrolyzed by purified PON3. PON1 efficiently hydrolyzed 5,6-DHTL, but with a specific activity about 15-fold lower than PON3. 5,6-DHTL was a poor substrate for PON2. Cyclo-EC was a poor substrate for PON1 and not hydrolyzed by PON2. Studies with the PON inhibitor EDTA and a serine esterase inhibitor indicated that the PONs are the main contributors to hydrolysis of the lactones in human and mouse liver homogenates. Studies with homogenates from PON3 knockout mouse livers indicated that >80% of the 5,6-DHTL and cyclo-EC lactonase activities were attributed to PON3. The findings provide further insight into the structural requirements for PONs substrates and support the hypothesis that PONs, particularly PON1 and PON3, evolved to hydrolyze and regulate a class of lactone lipid mediators derived from polyunsaturated fatty acids.


Assuntos
Arildialquilfosfatase/metabolismo , Eicosanoides/metabolismo , Lactonas/metabolismo , Animais , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Arildialquilfosfatase/genética , Eicosanoides/química , Células HEK293 , Humanos , Hidrólise , Lactonas/química , Fígado/metabolismo , Camundongos Knockout , Estrutura Molecular , Especificidade por Substrato
12.
Angew Chem Int Ed Engl ; 57(40): 13339-13343, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30048020

RESUMO

Lipid messengers exert their function on short time scales at distinct subcellular locations, yet most experimental approaches for perturbing their levels trigger cell-wide concentration changes. Herein, we report on a coumarin-based photocaging group that can be modified with organelle-targeting moieties by click chemistry and thus enables photorelease of lipid messengers in distinct organelles. We show that caged arachidonic acid and sphingosine derivatives can be selectively delivered to mitochondria, the ER, lysosomes, and the plasma membrane. By comparing the cellular calcium transients induced by localized uncaging of arachidonic acid and sphingosine, we show that the precise intracellular localization of the released second messenger is crucial for the signaling outcome. Ultimately, we anticipate that this new class of caged compounds will greatly facilitate the study of cellular processes on the organelle level.


Assuntos
Ácido Araquidônico/química , Química Click , Cumarínicos/química , Organelas/metabolismo , Esfingosina/análogos & derivados , Ácido Araquidônico/metabolismo , Cálcio/metabolismo , Cumarínicos/metabolismo , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Esfingosina/metabolismo , Imagem com Lapso de Tempo , Raios Ultravioleta
13.
Spectrochim Acta A Mol Biomol Spectrosc ; 203: 263-272, 2018 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-29879641

RESUMO

Arachidonic acid (AA) (cis,cis,cis,cis-5,8,11,14-Eicosatetraenoic acid) is an omega-6 polyunsaturated fatty acid (PUFA) constituent of the phospholipids of cell membranes. The conformational behavior of AA in the gas phase was investigated by means of density functional theory (DFT) using B3LYP method with 6-311++G(d,p) basis set. Theoretical calculations on the structures and infrared spectra of monomer conformers and dimer form of the most stable monomer conformer of AA were performed. Vibrational assignment of the fundamental modes was made based on calculated potential energy distribution (PED). Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectrum of AA in liquid phase was recorded in the region 4000-450 cm-1. The theoretical spectrum of dimer AA in gas phase is in reasonably good agreement with the experimental liquid phase spectrum. The double bonds in unsaturated fatty acids are prone to oxidation. Oxidized PUFAs lead to adverse health effects. The effects of daylight and temperature on the oxidative stability of AA were investigated using ATR-FTIR spectroscopy. The analysis reveals that the light and thermal treatment induce cis-trans isomerization in AA.


Assuntos
Ácido Araquidônico/química , Luz , Conformação Molecular , Temperatura Ambiente , Vibração , Espectroscopia de Infravermelho com Transformada de Fourier
14.
Methods Enzymol ; 605: 33-49, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29909831

RESUMO

Methods are presented for the use of the coral 8R-lipoxygenase from the Caribbean sea whip coral Plexaura homomalla as a model enzyme for structural studies of animal lipoxygenases. The 8R-lipoxygenase is remarkably stable and can be stored at 4°C for 3 months with virtually no loss of activity. In addition, an engineered "pseudo wild-type" enzyme is soluble in the absence of detergents, which helps facilitate the preparation of enzyme:substrate complexes.


Assuntos
Antozoários/metabolismo , Araquidonato Lipoxigenases/isolamento & purificação , Ácido Araquidônico/metabolismo , Ensaios Enzimáticos/métodos , Domínios Proteicos/genética , Animais , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/genética , Araquidonato Lipoxigenases/metabolismo , Ácido Araquidônico/química , Sítios de Ligação/genética , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/genética
15.
Methods Enzymol ; 605: 51-68, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29909837

RESUMO

Catalase-related allene oxide synthase (cAOS) is a hemoprotein that converts a specific fatty acid hydroperoxide to an unstable allene oxide intermediate at turnover rates in the order of 1000 per second. Fatty acid allene oxides are intermediates in the formation of cyclopentenone or hydrolytic products in marine systems, most notably the prostanoid-related clavulones. Although the key catalytic amino acid residues around the active site of cAOS are the same as in true catalases, cAOS does not react with hydrogen peroxide. cAOS occurs exclusively as the N-terminal domain of a naturally occurring fusion protein with a C-terminal lipoxygenase (LOX) domain that supplies the hydroperoxide substrate. In marine invertebrates, an 8R-LOX domain converts arachidonic acid to 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) and the cAOS domain forms an 8,9-epoxy allene oxide. The fusion protein from the sea whip octocoral Plexaura homomalla is the prototypical model with crystal structures of the individual domains. The cAOS (43kDa) expresses exceptionally well in Escherichia coli, with yields of up to 100mg/L. This article describes in detail expression and assay of the P. homomalla cAOS and two methods for the preparation of its 8R-HPETE substrate. Another article in this volume focuses on the P. homomalla 8R-LOX (Gilbert, Neau, & Newcomer, 2018).


Assuntos
Antozoários/metabolismo , Ensaios Enzimáticos/métodos , Hemeproteínas/metabolismo , Leucotrienos/síntese química , Lipoxigenase/metabolismo , Peroxidases/metabolismo , Animais , Ácido Araquidônico/química , Domínio Catalítico/genética , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Ciclopentanos/metabolismo , Escherichia coli/metabolismo , Hemeproteínas/genética , Hemeproteínas/isolamento & purificação , Peróxido de Hidrogênio/química , Leucotrienos/metabolismo , Lipoxigenase/genética , Lipoxigenase/isolamento & purificação , Oxirredução , Peroxidases/genética , Peroxidases/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
16.
Org Lett ; 20(13): 4020-4022, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29916257

RESUMO

The total synthesis of hemiketal E2 (HKE2) has been accomplished using a gold(I)-mediated cycloisomerization followed by oxidation of the enol ether product to introduce a unique keto-hemiketal, the core structure of HKE2. Synthetic hemiketal E2 reproduced biosynthetically derived HKE2 in the inhibition of human platelet aggregation.


Assuntos
Ácido Araquidônico/química , Plaquetas , Humanos , Estrutura Molecular , Oxirredução
17.
J Membr Biol ; 251(3): 475-489, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29610947

RESUMO

Unsaturated fatty acids are found in humans predominantly in the cis configuration. Fatty acids in the trans configuration are primarily the result of human processing (trans fats), but can also be formed endogenously by radical stress. The cis-trans isomerization of fatty acids by free radicals could be connected to several pathologies. Trans fats have been linked to an increased risk of coronary artery disease; however, the reasons for the resulting pathogenesis remain unclear. Here, we investigate the effect of a mono-trans isomer of arachidonic acid (C20:4-5trans, 8cis, 11cis, 14cis) produced by free radicals in physiological concentration on a model erythrocyte membrane using a combined experimental and theoretical approach. Molecular Dynamics (MD) simulations of two model lipid bilayers containing arachidonic acid and its 5-trans isomer in 3 mol% were carried out for this purpose. The 5-trans isomer formation in the phospholipids was catalyzed by HOCH2CH2S· radicals, generated from the corresponding thiol by γ-irradiation, in multilamellar vesicles of SAPC. Large unilamellar vesicles were made by the extrusion method (LUVET) as a biomimetic model for cis-trans isomerization. Atomic Force Microscopy and Dynamic Light Scattering were used to measure the average size, morphology, and the z-potential of the liposomes. Both results from MD simulations and experiments are in agreement and indicate that the two model membranes display different physicochemical properties in that the bilayers containing the trans fatty acids were more ordered and more rigid than those containing solely the cis arachidonic acid. Correspondingly, the average size of the liposomes containing trans isomers was smaller than the ones without.


Assuntos
Ácido Araquidônico/química , Lipossomos/química , Fosfolipídeos/química , Ácidos Graxos/química , Simulação de Dinâmica Molecular
18.
PLoS One ; 13(4): e0196398, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29698447

RESUMO

Drugs that inhibit cyclooxygenase (COX)-2 and the metabolism of arachidonic acid (ARA) to prostaglandin E2 are potent anti-inflammatory agents used widely in the treatment of joint and muscle pain. Despite their benefits, daily use of these drugs has been associated with hypertension, cardiovascular and gastrointestinal toxicities. It is now recognized that ARA is metabolized to a number of bioactive oxygenated lipids (oxylipins) by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP450) enzymes. Currently, the contribution of individual variability in ARA metabolism in response to the COX-2 inhibitors and potential adverse effects remains poorly understood. Using patient samples from the randomized, placebo-controlled phase III selenium/celecoxib (Sel/Cel) trial for the prevention of colorectal adenomatous polyps, we analyzed plasma concentrations of 74 oxylipins in a subset of participants who received celecoxib (n = 90) or placebo (n = 95). We assessed the effect of celecoxib (with and without low dose aspirin) on circulating oxylipins and systolic blood pressure (SBP). Individual CYP450- and LOX- but not COX-derived metabolites were higher with celecoxib than placebo (P<0.05) and differences were greater among non-aspirin users. LOX derived 5- and 8-HETE were elevated with celecoxib and positively associated with systolic blood pressure (P = 0.011 and P = 0.019 respectively). 20-HETE, a prohypertensive androgen-sensitive CYP450 metabolite was higher with celecoxib absent aspirin and was positively associated with SBP in men (P = 0.040) but not women. Independent of celecoxib or aspirin, LOX derived metabolites from ARA were strongly associated with SBP including 5- and 8-HETE. These findings support oxylipins, particularly the ARA LOX-derived, in blood pressure control and indicate that pharmacologic inhibition of COX-2 has effects on LOX and CYP450 ARA metabolism that contribute to hypertension in some patients.


Assuntos
Pólipos Adenomatosos/prevenção & controle , Celecoxib/uso terapêutico , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Oxilipinas/sangue , Pólipos/prevenção & controle , Pólipos Adenomatosos/patologia , Idoso , Anti-Inflamatórios não Esteroides/uso terapêutico , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Aspirina/uso terapêutico , Pressão Sanguínea , Celecoxib/metabolismo , Colo/patologia , Método Duplo-Cego , Ácidos Graxos Ômega-3/química , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/química , Ácidos Graxos Ômega-6/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Efeito Placebo , Pólipos/patologia , Selênio/uso terapêutico
20.
Comput Biol Chem ; 74: 1-11, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29522918

RESUMO

Soluble epoxide hydrolase (sEH), a key enzyme belonging to cytochrome P450 pathway of arachidonic acid cascade is a novel therapeutic drug target against atherosclerosis. The enzyme breaks down epoxyeicosatrienoic acid (EETs) to dihydroxy-eicosatrienoic acids (DHETs) and reduces beneficial cardiovascular properties of EETs. Thus, the present work is aimed at identification of potential leads as sEH inhibitors which will sustain the beneficial properties of EETs in vivo. PubChem and ZINC databases were screened for drug-like compounds based on Lipinski's rule of five and in silico toxicity filters. The binding potential of the drug-like compounds with sEH was explored using molecular docking. The top ranked lead (ZINC23099069) showed higher GOLD score compared with that of the control, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) and displayed two hydrogen bonds with Tyr383 and His420 and eleven residues involved in hydrophobic interactions with sEH. The apo_sEH and sEH_ZINC23099069 complex showed stable trajectories during 20 ns time scale of molecular dynamics (MD) simulation. Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) binding free energy analysis showed that electrostatic energy is the driving energy component for interaction of the lead with sEH. These results demonstrate ZINC23099069 to be a promising drug candidate as sEH inhibitor against atherosclerosis instead of the present urea-based inhibitors.


Assuntos
Ácido Araquidônico/farmacologia , Aterosclerose/tratamento farmacológico , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Ácido Araquidônico/síntese química , Ácido Araquidônico/química , Aterosclerose/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Epóxido Hidrolases/metabolismo , Humanos , Simulação de Dinâmica Molecular , Solubilidade , Termodinâmica
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