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1.
PLoS One ; 15(10): e0240189, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031441

RESUMO

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.


Assuntos
Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Tromboplastina/metabolismo , Coagulação Sanguínea , Plaquetas/metabolismo , Linhagem Celular , Humanos , Lipossomos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Tromboplastina/genética
2.
Proc Natl Acad Sci U S A ; 117(44): 27319-27328, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33087576

RESUMO

The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown. We found that spontaneous preterm birth is associated with ferroptosis and that inhibition of GPX4 causes ferroptotic injury in primary human trophoblasts and during mouse pregnancy. Importantly, we uncovered a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolize Hp-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia/reoxygenation injury in vivo. Together, we identified ferroptosis signaling in the human and mouse placenta, established a role for PLA2G6 in attenuating trophoblastic ferroptosis, and provided mechanistic insights into the ill-defined placental lipotoxicity that may inspire PLA2G6-targeted therapeutic strategies.


Assuntos
Ferroptose/fisiologia , Fosfolipases A2 do Grupo VI/metabolismo , Trofoblastos/metabolismo , Animais , Feminino , Glutationa Peroxidase/metabolismo , Fosfolipases A2 do Grupo VI/genética , Fosfolipases A2 do Grupo VI/fisiologia , Humanos , Ferro/metabolismo , Peróxidos Lipídicos/metabolismo , Camundongos , Camundongos Knockout , Fosfatidiletanolaminas/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Placenta/metabolismo , Gravidez , Nascimento Prematuro/metabolismo , Transdução de Sinais
3.
Nat Commun ; 11(1): 4317, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859896

RESUMO

Lipid membranes, nucleic acids, proteins, and metabolism are essential for modern cellular life. Synthetic systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced production of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of the activity of specific genes combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide experimental evidence for DNA-programmed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward effective liposome growth and self-reproduction.


Assuntos
Lipossomos/metabolismo , Lipídeos de Membrana/biossíntese , Lipídeos de Membrana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceróis/genética , Fosfatidilgliceróis/metabolismo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Proteômica
4.
Food Chem ; 332: 127384, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32615384

RESUMO

Dairy polar lipids (PL) seem to exhibit antiplatelet effects. However, it is not known what molecular species may be responsible. In this study, we confirmed using C30 reversed-phase (C30RP) ultra-high-performance liquid chromatography (UHPLC) coupled to high resolution accurate mass tandem mass spectrometry (HRAM-MS/MS) that fermentation of yoghurts from ovine milk using specific starter cultures altered the PL composition. These lipid alterations occurred concomitant with increased antithrombotic properties of the yoghurts PL fractions against platelet-activating factor (PAF) and thrombin-induced platelet aggregation. Specifically, elevation in phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylcholine (PC) and their molecular species were observed following yoghurt fermentation. Furthermore, PC(18:0/18:1), PE(18:1/18:2), SM(d18:0/22:0) and several other molecular species were significantly inversely correlated with the inhibition of PAF and thrombin. These molecular species were abundant in the most bioactive yoghurts fermented by S. thermophilus and L. acidophilus, which suggest that fermentation by these microorganisms increases the antithrombotic properties of ovine milk PL.


Assuntos
Lipídeos/análise , Leite/metabolismo , Inibidores da Agregação de Plaquetas/análise , Iogurte/análise , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cromatografia Líquida de Alta Pressão , Fermentação , Lipídeos/farmacologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Ovinos , Esfingomielinas/metabolismo , Espectrometria de Massas em Tandem , Trombina/farmacologia
5.
J Dairy Sci ; 103(9): 7742-7751, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32622597

RESUMO

Human colostrum is the first milk secreted by the mother after birth and constitutes the ideal food for the newborn, because its chemical composition, rich in immunoglobulins, antimicrobial peptides, growth factors, bioactive lipids, and other important molecules, is perfectly adapted to the metabolic, digestive, and immunological immaturity of the newborn. An incomplete gestational period can affect the maturity of the mammary gland and its ability to secrete milk with the proper composition for the newborn's condition. Previous studies indicate that the mammary gland modulates the profiles of bioactive lipids present in the different phases of lactation from colostrum to mature milk. Given the key role played by the polar lipids (PL) (phospho- and sphingolipids) of the milk fat globule membrane (MFGM) in the immune system and cognitive development of the newborn, it is crucial to analyze whether the content and distribution of the PL are affected by gestation period. Therefore, this study aimed to determine the milk fat globule (MFG) and MFGM lipid compositions of human colostrum samples from 20 healthy preterm and full-term mothers. Lipid characterization using chromatographic techniques (gas chromatograph mass spectrometry and HPLC-evaporative light-scattering detection) revealed differences related to length of gestation in the profiles of lipid classes and fatty acid and triacylglyceride contents of colostrum. This comparative analysis leads to noteworthy outcomes about the changing roles of the PL, considering the preterm or full-term condition. We found a lack of correlation of some PL (such as phosphatidylcholine, phosphatidylinositol, and phosphatidylserine) with the delivery term; these could be denoted as structural category lipids. However, sphingomyelin and phosphatidyl-ethanolamine exhibited trends to decrease in full-term colostrum, indicating that in the final stage of pregnancy specific accretion of some PL occurs, which should be denoted as a nutritional redistribution.


Assuntos
Colostro/química , Idade Gestacional , Glicolipídeos/química , Glicoproteínas/química , Gotículas Lipídicas/química , Leite Humano/química , Cromatografia Líquida de Alta Pressão/veterinária , Ácidos Graxos/análise , Feminino , Humanos , Recém-Nascido , Lactação , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Gravidez , Esfingomielinas/metabolismo
6.
Sci Rep ; 10(1): 12165, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699277

RESUMO

Macular Telangiectasia type 2 (MacTel) is an uncommon bilateral retinal disease, in which glial cell and photoreceptor degeneration leads to central vision loss. The causative disease mechanism is largely unknown, and no treatment is currently available. A previous study found variants in genes associated with glycine-serine metabolism (PSPH, PHGDH and CPS1) to be associated with MacTel, and showed low levels of glycine and serine in the serum of MacTel patients. Recently, a causative role of deoxysphingolipids in MacTel disease has been established. However, little is known about possible other metabolic dysregulation. Here we used a global metabolomics platform in a case-control study to comprehensively profile serum from 60 MacTel patients and 58 controls. Analysis of the data, using innovative computational approaches, revealed a detailed, disease-associated metabolic profile with broad changes in multiple metabolic pathways. This included alterations in the levels of several metabolites that are directly or indirectly linked to glycine-serine metabolism, further validating our previous genetic findings. We also found changes unrelated to PSPH, PHGDH and CPS1 activity. Most pronounced, levels of several lipid groups were altered, with increased phosphatidylethanolamines being the most affected lipid group. Assessing correlations between different metabolites across our samples revealed putative functional connections. Correlations between phosphatidylethanolamines and sphingomyelin, and glycine-serine and sphingomyelin, observed in controls, were reduced in MacTel patients, suggesting metabolic re-wiring of sphingomyelin metabolism in MacTel patients. Our findings provide novel insights into metabolic changes associated with MacTel and implicate altered lipid metabolism as a contributor to this retinal neurodegenerative disease.


Assuntos
Fosfatidiletanolaminas/sangue , Doenças Retinianas/patologia , Esfingomielinas/sangue , Idoso , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/etiologia , Feminino , Variação Genética , Glicina/metabolismo , Humanos , Masculino , Redes e Vias Metabólicas/genética , Metaboloma , Metabolômica/métodos , Pessoa de Meia-Idade , Fosfatidiletanolaminas/metabolismo , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Doenças Retinianas/complicações , Doenças Retinianas/metabolismo , Fatores de Risco , Serina/metabolismo , Esfingomielinas/metabolismo
7.
Sci Rep ; 10(1): 12299, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32704146

RESUMO

Milk fat globule (MFG) size is a milk production trait characteristic to the individual animal and has important effects on the functional and nutritional properties of milk. Although the regulation of MFG size in the mammary epithelial cell is not fully understood, lipid droplet (LD) fusion prior to secretion is believed to play a role. We selected cows that consistently produced milk with predominantly small or large MFGs to compare their lipidomic profiles, with focus on the polar lipid fraction. The polar lipid composition of the monolayer surrounding the LD is believed to either promote or prevent LD fusion. Using a targeted LC-MS/MS approach we studied the relative abundance of 301 detected species and found significant differences between the studied groups. Here we show that the lipidomic profile of milk from small MFG cows is characterised by higher phosphatidylcholine to phosphatidylethanolamine ratios. In contrast, the milk from large MFG cows contained more ether-phosphatidylethanolamine species. This is the first time that a potential role for ether-phosphatidylethanolamine in MFG size development has been suggested.


Assuntos
Células Epiteliais/metabolismo , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Gotículas Lipídicas/metabolismo , Glândulas Mamárias Animais/metabolismo , Fosfatidiletanolaminas/metabolismo , Característica Quantitativa Herdável , Animais , Bovinos , Cromatografia Líquida , Feminino , Metabolismo dos Lipídeos , Lipidômica/métodos , Redes e Vias Metabólicas , Espectrometria de Massas em Tandem
8.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641477

RESUMO

Positive-strand RNA [(+)RNA] viruses are important pathogens of humans, animals, and plants and replicate inside host cells by coopting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and coopted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with endoplasmic reticulum (ER)-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the double-stranded RNA (dsRNA) replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (-) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by phosphatidylinositol-3-phosphate [PI(3)P] and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 coopted replicase assembly factors, Vps34 phosphatidylinositol 3-kinase (PI3K) and the membrane-bending ESCRT factors, are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential coopted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication.IMPORTANCE Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals, and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and coopt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, we have developed an artificial giant unilamellar vesicle (GUV)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine, in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential coopted cellular factors.


Assuntos
RNA Replicase/genética , RNA de Cadeia Dupla/genética , Tombusvirus/genética , Lipossomas Unilamelares/metabolismo , Proteínas Virais/genética , Bioensaio , Linhagem Celular , Retículo Endoplasmático/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ergosterol/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , RNA Replicase/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Lipossomas Unilamelares/química , Proteínas Virais/metabolismo , Replicação Viral
9.
Curr Protoc Protein Sci ; 101(1): e110, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32603530

RESUMO

Peripheral membrane proteins participate in numerous biological pathways. Thus, methods to analyze their membrane-binding characteristics have become important. In this report, we detail protocols for the synthesis and utilization of a photoactivable fluorescent lipid as a reporter to monitor membrane binding of proteins. The assay, referred to as proximity-based labeling of membrane-associated proteins (PLiMAP), is based on UV activation of a fluorescent lipid reporter, which in turn crosslinks with proteins bound to membranes and renders them fluorescent. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of BODIPY-diazirine phosphatidylethanolamine (BDPE) Basic Protocol 2: Preparation of BDPE-containing liposomes Basic Protocol 3: Performing PLiMAP with a candidate protein Basic Protocol 4: Quantitation of liposome-binding properties of the candidate protein from analyzing in-gel fluorescence Support Protocol: Purification of GST-2×P4M domain of SidM protein.


Assuntos
Compostos de Boro/química , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Proteínas de Membrana/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Membrana Celular/química , Diazometano/química , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/química , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Processos Fotoquímicos , Ligação Proteica
10.
Planta ; 252(1): 4, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32524208

RESUMO

MAIN CONCLUSIONS: The main source of polyunsaturated acyl-CoA in cytoplasmic acyl-CoA pool of Camelina sativa seeds are fatty acids derived from phosphatidylcholine followed by phosphatidic acid. Contribution of phosphatidylethanolamine is negligible. While phosphatidylethanolamine (PE) is the second most abundant phospholipid, phosphatidic acid (PA) only constitutes a small fraction of C. sativa seeds' polar lipids. In spite of this, the relative contribution of PA in providing fatty acids for the synthesis of acyl-CoA, supplying cytosolic acyl-CoA pool seems to be much higher than the contribution of PE. Our data indicate that up to 5% of fatty acids present in mature C. sativa seeds are first esterified with PA, in comparison to 2% first esterified with PE, before being transferred into acyl-CoA pool via backward reactions of either acyl-CoA:lysophosphatidic acid acyltransferases (CsLPAATs) or acyl-CoA:lysophoshatidylethanolamine acyltransferases (CsLPEATs). Those acyl-CoAs are later reused for lipid biosynthesis or remodelling. In the forward reactions both aforementioned acyltransferases display the highest activity at 30 °C. The spectrum of optimal pH differs for both enzymes with CsLPAATs most active between pH 7.5-9.0 and CsLPEATs between pH 9.0 to 10.0. Whereas addition of magnesium ions stimulates CsLPAATs, calcium and potassium ions inhibit them in concentrations of 0.05-2.0 mM. All three types of ions inhibit CsLPEATs activity. Both tested acyltransferases present the highest preferences towards 16:0-CoA and unsaturated 18-carbon acyl-CoAs in forward reactions. However, CsLPAATs preferentially utilise 18:1-CoA and CsLPEATs preferentially utilise 18:2-CoA while catalysing fatty acid remodelling of PA and PE, respectively.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Camellia/enzimologia , Ácidos Fosfatídicos/metabolismo , Fosfatidiletanolaminas/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Acil Coenzima A/metabolismo , Camellia/genética , Camellia/crescimento & desenvolvimento , Ácidos Graxos/metabolismo , Lisofosfolipídeos/metabolismo , Fosfatidilcolinas/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento
11.
Proc Natl Acad Sci U S A ; 117(25): 14376-14385, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513718

RESUMO

Temporally harmonized elimination of damaged or unnecessary organelles and cells is a prerequisite of health. Under Type 2 inflammatory conditions, human airway epithelial cells (HAECs) generate proferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamines (HpETE-PEs) as proximate death signals. Production of 15-HpETE-PE depends on activation of 15-lipoxygenase-1 (15LO1) in complex with PE-binding protein-1 (PEBP1). We hypothesized that cellular membrane damage induced by these proferroptotic phospholipids triggers compensatory prosurvival pathways, and in particular autophagic pathways, to prevent cell elimination through programmed death. We discovered that PEBP1 is pivotal to driving dynamic interactions with both proferroptotic 15LO1 and the autophagic protein microtubule-associated light chain-3 (LC3). Further, the 15LO1-PEBP1-generated ferroptotic phospholipid, 15-HpETE-PE, promoted LC3-I lipidation to stimulate autophagy. This concurrent activation of autophagy protects cells from ferroptotic death and release of mitochondrial DNA. Similar findings are observed in Type 2 Hi asthma, where high levels of both 15LO1-PEBP1 and LC3-II are seen in HAECs, in association with low bronchoalveolar lavage fluid mitochondrial DNA and more severe disease. The concomitant activation of ferroptosis and autophagy by 15LO1-PEBP1 complexes and their hydroperoxy-phospholipids reveals a pathobiologic pathway relevant to asthma and amenable to therapeutic targeting.


Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Asma/imunologia , Autofagia/imunologia , Células Epiteliais/patologia , Ferroptose/imunologia , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Adulto , Animais , Asma/diagnóstico , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , Sobrevivência Celular/imunologia , Células Epiteliais/imunologia , Feminino , Técnicas de Inativação de Genes , Humanos , Ácidos Hidroxieicosatetraenoicos/imunologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Interleucina-13/imunologia , Interleucina-13/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Simulação de Dinâmica Molecular , Proteína de Ligação a Fosfatidiletanolamina/genética , Fosfatidiletanolaminas/imunologia , Fosfatidiletanolaminas/metabolismo , Cultura Primária de Células , Ligação Proteica/imunologia , Índice de Gravidade de Doença
12.
J Biol Chem ; 295(27): 9211-9222, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32430397

RESUMO

Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/ß-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.


Assuntos
Carboxiliases/análise , Corantes Fluorescentes/síntese química , Espectrometria de Fluorescência/métodos , Acetofenonas/química , Candida albicans/metabolismo , Carboxiliases/metabolismo , Membrana Celular/metabolismo , Etanolamina , Fluorescência , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mercaptoetanol/química , Mitocôndrias , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Estirenos/química
13.
Nat Chem Biol ; 16(6): 667-675, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393901

RESUMO

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.


Assuntos
Comportamento Animal/efeitos dos fármacos , Inibidores Enzimáticos/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Fosfatidiletanolaminas/metabolismo , Fosfolipase D/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Antagonistas de Receptores de Canabinoides/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Medo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Receptores de Canabinoides/metabolismo , Transdução de Sinais
14.
Biochim Biophys Acta Biomembr ; 1862(10): 183345, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32407777

RESUMO

Anabaena Sensory Rhodopsin (ASR) is a microbial photosensor from the cyanobacterium Anabaena sp. PCC 7120. It was found in previous studies that ASR co-purifies with several small molecules, although their identities and structural or functional roles remained unclear. Here, we use solid-state nuclear magnetic resonance (SSNMR) spectroscopy and mass spectrometry to characterize these molecules. Numerous correlations atypical for protein amino acids were found and assigned in the SSNMR spectra. The chemical shift patterns correspond to N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose which are part of the Enterobacterial Common Antigen (ECA). These sugars undergo rapid anisotropic motions and are likely linked flexibly to a rigid anchor that tightly binds ASR. Phosphorus NMR reveals several signals that are characteristic of monophosphates, further suggesting phosphatidylglyceride as the ECA lipid carrier which is anchored to ASR. In addition, NMR signals corresponding to common phospholipid phosphatidylethanolamine (PE) have been detected. The presence of PE tightly interacting with ASR was confirmed using liquid chromatography-mass spectrometry. This article commemorates Professor Michèle Auger and her contributions to membrane biophysics and Nuclear Magnetic Resonance.


Assuntos
Proteínas de Membrana/metabolismo , Fosfatidiletanolaminas/metabolismo , Rodopsinas Sensoriais/metabolismo , Anabaena/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Rodopsinas Sensoriais/química
15.
PLoS One ; 15(4): e0231289, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287294

RESUMO

Breast cancer (BC) is the most prevalent type of cancer in women in western countries. BC mortality has not declined despite early detection by screening, indicating the need for better informed treatment decisions. Therefore, a novel noninvasive diagnostic tool for BC would give the opportunity of subtype-specific treatment and improved prospects for the patients. Heterogeneity of BC tumor subtypes is reflected in the expression levels of enzymes in lipid metabolism. The aim of the study was to investigate whether the subtype defined by the transcriptome is reflected in the lipidome of BC cell lines. A liquid chromatography mass spectrometry (LC-MS) platform was applied to analyze the lipidome of six cell lines derived from human BC cell lines representing different BC subtypes. We identified an increased abundance of triacylglycerols (TG) ≥ C-48 with moderate or multiple unsaturation in fatty acyl chains and down-regulated ether-phosphatidylethanolamines (PE) (C-34 to C-38) in cell lines representing estrogen receptor and progesterone receptor positive tumor subtypes. In a cell line representing HER2-overexpressing tumor subtype an elevated expression of TG (≤ C-46), phosphatidylcholines (PC) and PE containing short-chained (≤ C-16) saturated or monounsaturated fatty acids were observed. Increased abundance of PC ≥ C-40 was found in cell lines of triple negative BC subtype. In addition, differences were detected in lipidomes within these previously defined subtypes. We conclude that subtypes defined by the transcriptome are indeed reflected in differences in the lipidome and, furthermore, potentially biologically relevant differences may exist within these defined subtypes.


Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/metabolismo , Metabolismo dos Lipídeos , Lipidômica/métodos , Linhagem Celular Tumoral , Feminino , Humanos , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Triglicerídeos/metabolismo
16.
J Agric Food Chem ; 68(15): 4507-4514, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32223231

RESUMO

The objective of this study was to evaluate whether whole raw milk originating from Holstein dairy cows affected by lameness alters its composition. A total of 20 healthy control cows and 6 cows diagnosed with lameness were selected out of 100 sampled cows in a nested case control study at 2 weeks postpartum, and whole raw milk samples were collected and analyzed with direct inject/liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance. In total, 168 metabolites were identified and quantified using an in-house mass spectrometry library. A total of 35 of the identified metabolites decreased versus control cows. Only two metabolites (i.e., sn-glycero-3-phosphocholine and phosphatidylethanolamine ae C42:1) were increased in the milk of lame cows. In conclusion, milk metabotyping of lame cows revealed significant changes in multiple milk components, including amino acids, lipids, and biogenic amines. Most of the milk compounds identified as altered were lowered, suggesting deflection of nutrients from the mammary gland to the host needs for healing lameness-associated pathological processes.


Assuntos
Doenças dos Bovinos/metabolismo , Coxeadura Animal/metabolismo , Leite/química , Leite/metabolismo , Animais , Aminas Biogênicas/química , Aminas Biogênicas/metabolismo , Bovinos , Doenças dos Bovinos/fisiopatologia , Feminino , Glicerilfosforilcolina/química , Glicerilfosforilcolina/metabolismo , Lactação , Coxeadura Animal/fisiopatologia , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo
17.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32269127

RESUMO

Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication.IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.


Assuntos
Interações Hospedeiro-Patógeno/genética , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tombusvirus/genética , Replicação Viral/genética , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endossomos/metabolismo , Regulação da Expressão Gênica , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Protoplastos/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , RNA Replicase/genética , RNA Replicase/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Esteróis/metabolismo , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
18.
Biomed Res Int ; 2020: 5393041, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32149115

RESUMO

Resveratrol (Resv) offers health benefits in cancer and has been reported to modulate important enzymes of lipid metabolism. Studies of its effects on lipid composition in different subtypes of breast-cancer cells are scarce. Thus, we investigated the alterations in phospholipids (PL), fatty acids (FA), and lipid metabolism enzymes in two breast-cancer cell lines after Resv treatment. MCF-7 and MDA-MB-231 cells were treated with 80 and 200 µM of Resv, respectively, for 24 hours. We analyzed PL with radiolabeled inorganic phosphate (32Pi) by thin-layer chromatography, FA by gas chromatography-mass spectrometry, and lipid metabolism enzymes (DGAT2, FAS, ρACCß, pAMPKα, and AMPK) by Western blot. Resv treated MDA-MB-231 phospholipids showed a reduction in phosphatidylcholine (63%) and phosphatidylethanolamine (35%). We observed an increase in eicosapentaenoic acid (EPA) (73%) and docosahexaenoic acid (DHA) (65%) in MCF-7 cells after Resv treatment. Interestingly, the same treatment caused 50% and 90% increases in EPA and DHA, respectively, in MDA-MB-231 cells. In MCF-7 cells, Resv increased the expression of ρACCß (3.3-fold) and AMPKα/ρAMPKα (1.5-fold) and in MDA-MB-231 cells it inhibited the expression of ρACCß (111.8-fold) and AMPKα/ρAMPKα (1.2 fold). Our results show that Resv modified PL and saturated and unsaturated FA especially in MDA-MB-231 cells, and open new perspectives to the understanding of the reported anticancer effect of Resv on these cells.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Resveratrol/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados , Feminino , Humanos , Lipídeos/análise , Células MCF-7 , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolipídeos/metabolismo , Resveratrol/uso terapêutico
19.
J Biol Chem ; 295(13): 4124-4133, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221031

RESUMO

An early exposure to lipid biochemistry in the laboratory of Konrad Bloch resulted in a fascination with the biosynthesis, structures, and functions of bacterial lipids. The discovery of plasmalogens (1-alk-1'-enyl, 2-acyl phospholipids) in anaerobic Gram-positive bacteria led to studies on the physical chemistry of these lipids and the cellular regulation of membrane lipid polymorphism in bacteria. Later studies in several laboratories showed that the formation of the alk-1-enyl ether bond involves an aerobic process in animal cells and thus is fundamentally different from that in anaerobic organisms. Our work provides evidence for an anaerobic process in which plasmalogens are formed from their corresponding diacyl lipids. Studies on the roles of phospholipases in Listeria monocytogenes revealed distinctions between its phospholipases and those previously discovered in other bacteria and showed how the Listeria enzymes are uniquely fitted to the intracellular lifestyle of this significant human pathogen.


Assuntos
Anaerobiose/genética , Lipídeos/genética , Plasmalogênios/metabolismo , Bactérias Anaeróbias/genética , Bactérias Anaeróbias/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Lipídeos/biossíntese , Lipídeos/química , Fosfatidiletanolaminas/biossíntese , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Plasmalogênios/química , Plasmalogênios/genética
20.
Chem Biol Interact ; 319: 109019, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32092302

RESUMO

The inhibition of the enzyme acetylcholinesterase (AChE) is a frequently used therapeutic option to treat Alzheimer's disease (AD). By decreasing the levels of acetylcholine degradation in the synaptic space, some cognitive functions of patients suffering from this disease are significantly improved. Rivastigmine is one of the most widely used AChE inhibitors. The objective of this work was to determine the effects of this drug on human erythrocytes, which have a type of AChE in the cell membrane. To that end, human erythrocytes and molecular models of its membrane constituted by dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were used. They correspond to classes of phospholipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively. The experimental results obtained by X-ray diffraction and differential scanning calorimetry (DSC) indicated that rivastigmine molecules were able to interact with both phospholipids. Fluorescence spectroscopy results showed that rivastigmine produce a slight change in the acyl chain packing order and a weak displacement of the water molecules of the hydrophobic-hydrophilic membrane interface. On the other hand, observations by scanning electron microscopy (SEM) showed that the drug changed the normal biconcave shape of erythrocytes in stomatocytes (cup-shaped cells) and echinocytes (speculated shaped).


Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores da Colinesterase/farmacologia , Eritrócitos/efeitos dos fármacos , Rivastigmina/farmacologia , Acetilcolinesterase/metabolismo , Doença de Alzheimer/metabolismo , Varredura Diferencial de Calorimetria/métodos , Forma Celular/efeitos dos fármacos , Dimiristoilfosfatidilcolina/metabolismo , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Humanos , Microscopia Eletrônica de Varredura/métodos , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Fosfolipídeos/metabolismo , Espectrometria de Fluorescência/métodos , Difração de Raios X/métodos
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