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1.
Mol Cell ; 75(5): 1043-1057.e8, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31402097

RESUMO

The plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet, how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP-related Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Biophysical FRET analyses suggest an unconventional co-distribution of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, using in vivo imaging approaches and molecular dynamics simulations, we show that Osh protein-mediated unsaturated PI4P and PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP family members create a nanoscale membrane lipid environment that drives PIP5K activity and PI(4,5)P2 synthesis that ultimately controls global PM organization and dynamics.

2.
J Mol Cell Cardiol ; 133: 1-11, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145942

RESUMO

BACKGROUND: The fatty acid (FA) composition of membrane phospholipid reflects at least in part dietary fat composition. Saturated FA (SFA) suppress Sirt1 activity, while monounsaturated FA (MUFA) counteract this effect. OBJECTIVE: We explored a role of Sirt1 in homeostatic control of the fatty acid composition of membrane phospholipid in the presence of SFA overload. METHODS AND RESULTS: Sirt1 deficiency in cardiomyocytes decreased the expression levels of liver X receptor (LXR)-target genes, particularly stearoyl-CoA desaturase-1 (Scd1), a rate-limiting enzyme in the cellular synthesis of MUFA from SFA, increased membrane SFA/MUFA ratio, and worsened left ventricular (LV) diastolic function in mice fed an SFA-rich high fat diet. In cultured cardiomyocytes, Sirt1 knockdown (KD) exacerbated the palmitate overload-induced increase in membrane SFA/MUFA ratio, which was associated with decrease in the expression of LXR-target genes, including Scd1. Forced overexpression of Scd1 in palmitate-overloaded Sirt1KD cardiomyocytes lowered the SFA/MUFA ratio. Nicotinamide mononucleotide (NMN) increased Sirt1 activity and Scd1 expression, thereby lowering membrane SFA/MUFA ratio in palmitate-overloaded cardiomyocytes. These effects of NMN were not observed for Scd1KD cardiomyocytes. LXRα/ßKD exacerbated palmitate overload-induced increase in membrane SFA/MUFA ratio, while LXR agonist T0901317 alleviated it. NMN failed to rescue Scd1 protein expression and membrane SFA/MUFA ratio in palmitate-overloaded LXRα/ßKD cardiomyocytes. The administration of NMN or T0901317 showed a dramatic reversal in membrane SFA/MUFA ratio and LV diastolic function in SFA-rich HFD-fed mice. CONCLUSION: Cardiac Sirt1 counteracted SFA overload-induced decrease in membrane phospholipid unsaturation and diastolic dysfunction via regulating LXR-mediated transcription of the Scd1 gene.

3.
Arch Biochem Biophys ; 663: 120-128, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30629958

RESUMO

BACKGROUND: Vitamin C (l-ascorbic acid, VC) and vitamin E (α-tocopherol, VE) play important physiological roles as endogenous antioxidants in many tissues and organs. However, their roles in the brain remain entirely elusive. We established senescence marker protein 30 (SMP30)/α-tocopherol transfer protein (αTTP) double knockout (DKO) mice as a novel VC and VE double-deficiency model and examined the effect of VC and VE double-deficiency on brain functions. METHODS: DKO and wild-type (WT) mice were divided into the following two groups: mice in the CE (+) group were supplied with sufficient amounts of VC and VE and mice in the CE (-) group were deficient in both VC and VE. After 8 weeks of CE (+) or CE (-) treatments, a battery of behavioral experiments was conducted to analyze cognitive functions, including memory, through the Morris water maze and Pavlovian fear conditioning tasks. RESULTS: The plasma VC and VE levels in DKO-CE (-) mice and VE level in WT-CE (-) mice were almost completely depleted after 8 weeks of the deficient treatment. The behavioral study revealed that the general behaviors, including locomotor activity and anxiety level, were not influenced by the CE (-) treatment in DKO and WT mice. However, in the Pavlovian fear conditioning task, DKO-CE (-) mice showed impaired conditioned fear memory compared with that of DKO-CE (+) mice. Furthermore, increased mRNA expression was observed in inflammatory-related genes, such as IL-6, TNFα, F4/80, and Mcp-1, in the hippocampus of DKO-CE (-) mice. CONCLUSIONS: The findings of this study provide evidence that VC and VE deficiency led to impaired conditioned fear memory possibly caused by neuroinflammation in the brain.

4.
PLoS One ; 13(12): e0208396, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30533011

RESUMO

Increase in saturated fatty acid (SFA) content in membrane phospholipids dramatically affects membrane properties and cellular functioning. We sought to determine whether exogenous SFA from the diet directly affects the degree of membrane phospholipid unsaturation in adult hearts and if these changes correlate with contractile dysfunction. Although both SFA-rich high fat diets (HFDs) and monounsaturated FA (MUFA)-rich HFDs cause the same degree of activation of myocardial FA uptake, triglyceride turnover, and mitochondrial FA oxidation and accumulation of toxic lipid intermediates, the former induced more severe diastolic dysfunction than the latter, which was accompanied with a decrease in membrane phospholipid unsaturation, induction of unfolded protein response (UPR), and a decrease in the expression of Sirt1 and stearoyl-CoA desaturase-1 (SCD1), catalyzing the conversion of SFA to MUFA. When the SFA supply in the heart overwhelms the cellular capacity to use it for energy, excess exogenous SFA channels to membrane phospholipids, leading to UPR induction, and development of diastolic dysfunction.


Assuntos
Cardiomiopatias/metabolismo , Lipídeos de Membrana/metabolismo , Membranas/metabolismo , Fosfolipídeos/metabolismo , Animais , Cardiomiopatias/patologia , Células Cultivadas , Diástole , Dieta Hiperlipídica , Regulação para Baixo , Ácidos Graxos Monoinsaturados/análise , Ácidos Graxos Monoinsaturados/metabolismo , Masculino , Lipídeos de Membrana/análise , Membranas/química , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/química , Miocárdio/metabolismo , Fosfolipídeos/análise , Triglicerídeos/análise , Triglicerídeos/metabolismo , Resposta a Proteínas não Dobradas/fisiologia
5.
Biochem Biophys Res Commun ; 505(1): 81-86, 2018 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-30241938

RESUMO

Reelin is a secreted protein essential for the development and function of the mammalian brain. The receptors for Reelin, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, belong to the low-density lipoprotein receptor family, but it is not known whether Reelin is involved in the brain lipid metabolism. In the present study, we performed lipidomic analysis of the cerebral cortex of wild-type and Reelin-deficient (reeler) mice, and found that reeler mice exhibited several compositional changes in phospholipids. First, the ratio of phospholipids containing one saturated fatty acid (FA) and one docosahexaenoic acid (DHA) or arachidonic acid (ARA) decreased. Secondly, the ratio of phospholipids containing one monounsaturated FA (MUFA) and one DHA or ARA increased. Thirdly, the ratio of phospholipids containing 5,8,11-eicosatrienoic acid, or Mead acid (MA), increased. Finally, the expression of stearoyl-CoA desaturase-1 (SCD-1) increased. As the increase of MA is seen as an index of polyunsaturated FA (PUFA) deficiency, and the expression of SCD-1 is suppressed by PUFA, these results strongly suggest that the loss of Reelin leads to PUFA deficiency. Hence, MUFA and MA are synthesized in response to this deficiency, in part by inducing SCD-1 expression. This is the first report of changes of FA composition in the reeler mouse brain and provides a basis for further investigating the new role of Reelin in the development and function of the brain.

6.
Artigo em Inglês | MEDLINE | ID: mdl-30055287

RESUMO

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique members of the phospholipase A2 family that can hydrolyze the acetyl group of PAF, a signaling phospholipid that has roles in diverse (patho)physiological processes. Three types of PAF-AH have been identified in mammals, one plasma type and two intracellular types [PAF-AH (I) and PAF-AH (II)]. Plasma PAF-AH and PAF-AH (II) are monomeric enzymes that are structurally similar, while PAF-AH (I) is a multimeric enzyme with no homology to other PAF-AHs. PAF-AH (I) shows a strong preference for an acetyl group, whereas plasma PAF-AH and PAF-AH (II) also hydrolyze phospholipids with oxidatively modified fatty acids. Plasma PAF-AH has been implicated in several diseases including cardiovascular disease. PAF-AH (I) is required for spermatogenesis and is increasingly recognized as an oncogenic factor. PAF-AH (II) was recently shown to act as a bioactive lipid-producing enzyme in mast cells and thus could be a drug target for allergic diseases. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.

7.
Nat Med ; 23(11): 1287-1297, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29035365

RESUMO

Critical to the function of mast cells in immune responses including allergy is their production of lipid mediators, among which only omega-6 (ω-6) arachidonate-derived eicosanoids have been well characterized. Here, by employing comprehensive lipidomics, we identify omega-3 (ω-3) fatty acid epoxides as new mast cell-derived lipid mediators and show that they are produced by PAF-AH2, an oxidized-phospholipid-selective phospholipase A2. Genetic or pharmacological deletion of PAF-AH2 reduced the steady-state production of ω-3 epoxides, leading to attenuated mast cell activation and anaphylaxis following FcɛRI cross-linking. Mechanistically, the ω-3 epoxides promote IgE-mediated activation of mast cells by downregulating Srcin1, a Src-inhibitory protein that counteracts FcɛRI signaling, through a pathway involving PPARg. Thus, the PAF-AH2-ω-3 epoxide-Srcin1 axis presents new potential drug targets for allergic diseases.


Assuntos
Compostos de Epóxi/química , Ácidos Graxos Ômega-3/farmacologia , Imunoglobulina E/imunologia , Mastócitos/imunologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Animais , Células Cultivadas , Meios de Cultivo Condicionados , Ácidos Graxos Ômega-3/química , Humanos , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fosfolipídeos/metabolismo
8.
Mol Biol Cell ; 28(17): 2318-2332, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28615323

RESUMO

Altered cellular lipid composition activates the endoplasmic reticulum unfolded protein response (UPR), and UPR signaling effects important changes in lipid metabolism. Secondary effects on protein folding homeostasis likely contribute to UPR activation, but deletion of the unfolded protein stress-sensing luminal domain of the UPR transducers PERK and IRE1α does not abolish their responsiveness to lipid perturbation. This finding suggests that PERK and IRE1α also directly recognize the membrane aberrancy wrought by lipid perturbation. However, beyond the need for a transmembrane domain (TMD), little is known about the features involved. Regulation of the UPR transducers entails changes in their oligomeric state and is easily corrupted by overexpression. We used CRISPR/Cas9-mediated gene editing of the Ern1 locus to study the role of the TMD in the ability of the endogenous IRE1α protein to recognize membrane aberrancy in mammalian cells. Conducted in the background of a point mutation that isolated the response to membrane aberrancy induced by palmitate from unfolded protein stress, our analysis shows that generic membrane-spanning features of the TMD are sufficient for IRE1α's responsiveness to membrane aberrancy. Our data suggest that IRE1α's conserved TMD may have been selected for features imparting a relatively muted response to acyl-chain saturation.


Assuntos
Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , eIF-2 Quinase/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/genética , Lipídeos , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Mutação , Ácido Palmítico , Domínios Proteicos , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo
9.
EMBO J ; 36(12): 1719-1735, 2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28495679

RESUMO

The autophagosome, a double-membrane structure mediating degradation of cytoplasmic materials by macroautophagy, is formed in close proximity to the endoplasmic reticulum (ER). However, how the ER membrane is involved in autophagy initiation and to which membrane structures the autophagy-initiation complex is localized have not been fully characterized. Here, we were able to biochemically analyze autophagic intermediate membranes and show that the autophagy-initiation complex containing ULK and FIP200 first associates with the ER membrane. To further characterize the ER subdomain, we screened phospholipid biosynthetic enzymes and found that the autophagy-initiation complex localizes to phosphatidylinositol synthase (PIS)-enriched ER subdomains. Then, the initiation complex translocates to the ATG9A-positive autophagosome precursors in a PI3P-dependent manner. Depletion of phosphatidylinositol (PI) by targeting bacterial PI-specific phospholipase C to the PIS domain impairs recruitment of downstream autophagy factors and autophagosome formation. These findings suggest that the autophagy-initiation complex, the PIS-enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.


Assuntos
Autofagossomos/metabolismo , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/análise , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Biogênese de Organelas , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Transporte Proteico
10.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(7): 658-665, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28373057

RESUMO

The ATP-binding cassette transporter A7 (ABCA7), which is highly expressed in the brain, is associated with the pathogenesis of Alzheimer's disease (AD). However, the physiological function of ABCA7 and its transport substrates remain unclear. Immunohistochemical analyses of human brain sections from AD and non-AD subjects revealed that ABCA7 is expressed in neuron and microglia cells in the cerebral cortex. The transport substrates and acceptors were identified in BHK/ABCA7 cells and compared with those of ABCA1. Like ABCA1, ABCA7 exported choline phospholipids in the presence of apoA-I and apoE; however, unlike ABCA1, cholesterol efflux was marginal. Lipid efflux by ABCA7 was saturated by 5µg/ml apoA-I and was not dependent on apoE isoforms, whereas efflux by ABCA1 was dependent on apoA-I up to 20µg/ml and apoE isoforms. Liquid chromatography-tandem mass spectrometry analyses revealed that the two proteins had different preferences for phospholipid export: ABCA7 preferred phosphatidylcholine (PC)≥lysoPC>sphingomyelin (SM)=phosphatidylethanolamine (PE), whereas ABCA1 preferred PC>>SM>PE=lysoPC. The major difference in the pattern of lipid peaks between ABCA7 and ABCA1 was the high lysoPC/PC ratio of ABCA7. These results suggest that lysoPC is one of the major transport substrates for ABCA7 and that lysoPC export may be a physiologically important function of ABCA7 in the brain.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Lisofosfatidilcolinas/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Doença de Alzheimer/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Colesterol/metabolismo , Cricetinae , Células HEK293 , Humanos , Metabolismo dos Lipídeos/fisiologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolipídeos/metabolismo , Esfingomielinas/metabolismo
11.
Mol Biol Cell ; 28(1): 161-172, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28035047

RESUMO

Phosphoinositides (PIPs) are key regulators of membrane traffic and signaling. The interconversion of PIPs by lipid kinases and phosphatases regulates their functionality. Phosphatidylinositol (PI) and PIPs have a unique enrichment of 1-stearoyl-2-arachidonyl acyl species; however, the regulation and function of this specific acyl profile remains poorly understood. We examined the role of the PI acyltransferase LYCAT in control of PIPs and PIP-dependent membrane traffic. LYCAT silencing selectively perturbed the levels and localization of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-3-phosphate and the membrane traffic dependent on these specific PIPs but was without effect on phosphatidylinositol-4-phosphate or biosynthetic membrane traffic. The acyl profile of PI(4,5)P2 was selectively altered in LYCAT-deficient cells, whereas LYCAT localized with phosphatidylinositol synthase. We propose that LYCAT remodels the acyl chains of PI, which is then channeled into PI(4,5)P2 Our observations suggest that the PIP acyl chain profile may exert broad control of cell physiology.


Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/fisiologia , Fosfatidilinositóis/metabolismo , Aciltransferases/metabolismo , Aciltransferases/fisiologia , Técnicas de Cultura de Células , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases , Transporte Proteico/fisiologia , Epitélio Pigmentado da Retina
12.
Eur J Nutr ; 56(3): 1317-1327, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26893162

RESUMO

PURPOSE: Despite numerous studies on the RRR- and all-rac-α-tocopherol isoform of vitamin E (VE) during aging, this relationship has not been examined in specific tissues. Since α-tocopherol is the most abundant of VE's eight isoforms, and VE is an important antioxidant that impacts the aging process, we analyzed α-tocopherol levels in plasma and tissues of mice at progressive ages. Moreover, we examined protein and mRNA expression levels of hepatic α-tocopherol transfer protein (α-TTP), which specifically binds α-tocopherol, during aging. METHODS: The α-tocopherol levels in plasma, liver, cerebrum, hippocampus, cerebellum, heart, kidney, epididymal adipose tissue, testis, pancreas, soleus muscle, plantaris muscle, and duodenum from male C57BL/6NCr mice at 3, 6, 12, 18, and 24 months of age were determined by HPLC and fluorescence detection. Also, hepatic α-TTP protein and mRNA expression levels were analyzed by Western blot and qPCR, respectively. RESULTS: Tissue-specific, age-related changes of α-tocopherol levels normalized by tissue weight were observed in the liver, cerebrum, hippocampus, cerebellum, heart, kidney, and epididymal adipose tissue. Specifically, α-tocopherol levels in epididymal adipose tissue increased greatly as mice aged from 6 to 24 months. Although hepatic α-TTP protein levels also showed age-related changes, α-TTP mRNA expression levels measured after overnight fasting were not altered. CONCLUSIONS: In this study, we determined that α-tocopherol levels and hepatic α-TTP protein levels of mice undergo significant tissue-specific, age-related changes. This is the first report to investigate VE in terms of the α-tocopherol levels in plasma and various tissues of mice and hepatic α-TTP protein levels during aging.


Assuntos
Envelhecimento , Proteínas de Transporte/metabolismo , Vitamina E/sangue , alfa-Tocoferol/sangue , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Antioxidantes/administração & dosagem , Peso Corporal , Proteínas de Transporte/genética , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Triglicerídeos/sangue , Vitamina E/administração & dosagem , alfa-Tocoferol/administração & dosagem
13.
PLoS Genet ; 12(8): e1006276, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27564576

RESUMO

Mg2+ serves as an essential cofactor for numerous enzymes and its levels are tightly regulated by various Mg2+ transporters. Here, we analyzed Caenorhabditis elegans strains carrying mutations in genes encoding cyclin M (CNNM) Mg2+ transporters. We isolated inactivating mutants for each of the five Caenorhabditis elegans cnnm family genes, cnnm-1 through cnnm-5. cnnm-1; cnnm-3 double mutant worms showed various phenotypes, among which the sterile phenotype was rescued by supplementing the media with Mg2+. This sterility was caused by a gonadogenesis defect with severely attenuated proliferation of germ cells. Using this gonadogenesis defect as an indicator, we performed genome-wide RNAi screening, to search for genes associated with this phenotype. The results revealed that RNAi-mediated inactivation of several genes restores gonad elongation, including aak-2, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK). We then generated triple mutant worms for cnnm-1; cnnm-3; aak-2 and confirmed that the aak-2 mutation also suppressed the defective gonadal elongation in cnnm-1; cnnm-3 mutant worms. AMPK is activated under low-energy conditions and plays a central role in regulating cellular metabolism to adapt to the energy status of cells. Thus, we provide genetic evidence linking Mg2+ homeostasis to energy metabolism via AMPK.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte de Cátions/genética , Ciclinas/genética , Longevidade/genética , Complexos Multiproteicos/genética , Proteínas Serina-Treonina Quinases/genética , Serina-Treonina Quinases TOR/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/metabolismo , Ciclinas/biossíntese , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/metabolismo , Gônadas/crescimento & desenvolvimento , Gônadas/metabolismo , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/metabolismo , Magnésio/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Família Multigênica/genética , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Transdução de Sinais/genética
14.
FASEB J ; 30(5): 2027-39, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26887439

RESUMO

The degree of fatty acid unsaturation in membrane phospholipids affects many membrane-associated functions and can be influenced by dietary consumption of fatty acids such as saturated fatty acids and polyunsaturated fatty acids (PUFAs). Cells must adapt to changes in composition of membrane fatty acids by regulating lipid-metabolizing enzymes. In this study, we investigated how cells respond to loading with excess PUFAs, such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. A lipidomics analysis revealed that dipalmitoylphosphatidylcholine (DPPC) was increased after the production of PUFA-containing phospholipids in cells loaded with PUFAs. An RNA interference screen of lipid-metabolizing enzymes revealed that lysophosphatidylcholine acyltransferase 1 (LPCAT1) was involved in the DPPC production. Moreover, LPCAT1 knockdown markedly enhanced the cytotoxicity induced by excess PUFAs. PUFA-induced cytotoxicity was dependent on caspase and unfolded protein response (UPR) sensor proteins inositol requiring 1α and protein kinase R-like endoplasmic reticulum kinase, suggesting that excess PUFAs trigger UPR-mediated apoptosis. In murine retina, in which PUFAs are highly enriched, DPPC was produced along with increase of PUFA-containing phospholipids. In LPCAT1 knockout mice, DPPC level was reduced and UPR was activated in the retina. Our results provide insight into understanding of the retinal degeneration seen in rd11 mice that lack LPCAT1.-Akagi, S., Kono, N., Ariyama, H., Shindou, H., Shimizu, T., Arai, H. Lysophosphatidylcholine acyltransferase 1 protects against cytotoxicity induced by polyunsaturated fatty acids.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Ácidos Graxos Insaturados/toxicidade , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Animais , Linhagem Celular , Sobrevivência Celular , Ácido Eicosapentaenoico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Knockout , Retina/metabolismo , Degeneração Retiniana/metabolismo
15.
Exp Cell Res ; 342(1): 1-10, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26896729

RESUMO

EHD3 is localized on the tubular structures of early endosomes, and it regulates their trafficking pathway. However, the regulatory mechanism of EHD3-containing tubular structures remains poorly understood. An in vitro liposome co-sedimentation assay revealed that EHD3 interacted with phosphatidic acid through its helical domain and this interaction induced liposomal tubulations. Additionally, inhibiting phosphatidic acid synthesis with diacylglycerol kinase inhibitor or lysophosphatidic acid acyltransferase inhibitor significantly reduced the number of EHD3-containing tubules and impaired their trafficking from early endosomes. These results suggest that EHD3 and phosphatidic acid cooperatively regulate membrane deformation and trafficking from early endosomes.


Assuntos
Proteínas de Transporte/metabolismo , Extensões da Superfície Celular/metabolismo , Ácidos Fosfatídicos/fisiologia , Sequência de Aminoácidos , Animais , Endocitose , Endossomos/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Vesículas Transportadoras/metabolismo
16.
Enzymes ; 38: 43-54, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26612646

RESUMO

Intracellular platelet-activating factor acetylhydrolase, type II [PAF-AH (II)] is a monomeric 40kDa enzyme that was originally purified as an enzyme that hydrolyzes the acetyl group of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). It can also hydrolyze phospholipids with short length or oxidatively modified sn-2 acyl chains, whereas it can hardly hydrolyze phospholipids with two long fatty acyl chains. PAF-AH (II) is the only phospholipase A2 that is myristoylated at its N-terminus and is evolutionarily conserved from lower organisms to mammals. Studies using cultured cells and mice suggest that PAF-AH (II) functions as an antioxidant phospholipase. Here, we will review our current understanding of PAF-AH (II) and discuss its potential functions in physiological and pathological conditions.

17.
J Lipid Res ; 56(10): 1880-90, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26239183

RESUMO

PUFAs, which account for 25-30% of the total fatty acids in the human brain, are important for normal brain development and cognitive function. However, it remains unclear how PUFAs are delivered to neurons and exert their effects. In this study, we demonstrated that n-3 and n-6 PUFAs added to the medium are incorporated into membrane phospholipids of primary glial cells from rat cortices, and then secreted as the fatty acid moiety of phospholipids in apoE-containing lipoproteins (LpEs). Tandem mass spectrometry analysis further showed that LpEs secreted from glial cells contain a variety of metabolites of PUFAs produced in glial cells by elongation and unsaturation. LpEs are absorbed by endocytosis into neurons via LDL receptor-related protein 1. LpE-containing n-3 and n-6 PUFAs exhibit a strong effect on neurite outgrowth of hippocampal neurons by increasing the number of branches. This study sheds light on the novel role of LpEs in the central nervous system and also a novel pathway in which PUFAs act on neurons.


Assuntos
Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Neuritos/fisiologia , Neuroglia/citologia , Animais , Apolipoproteínas E/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Meios de Cultura , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Hipocampo/metabolismo , Lipoproteínas/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Neuritos/metabolismo , Neuroglia/metabolismo , Fosfolipídeos/metabolismo , Ratos , Ratos Sprague-Dawley
18.
J Biol Chem ; 290(28): 17520-34, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-26018079

RESUMO

Phospholipase A/acyltransferase (PLA/AT)-3 (also known as H-rev107 or AdPLA) was originally isolated as a tumor suppressor and was later shown to have phospholipase A1/A2 activity. We have also found that the overexpression of PLA/AT-3 in mammalian cells results in specific disappearance of peroxisomes. However, its molecular mechanism remained unclear. In the present study, we first established a HEK293 cell line, which stably expresses a fluorescent peroxisome marker protein (DsRed2-Peroxi) and expresses PLA/AT-3 in a tetracycline-dependent manner. The treatment with tetracycline, as expected, caused disappearance of peroxisomes within 24 h, as revealed by diffuse signals of DsRed2-Peroxi and a remarkable decrease in a peroxisomal membrane protein, PMP70. A time-dependent decrease in ether-type lipid levels was also seen. Because the activation of LC3, a marker of autophagy, was not observed, the involvement of autophagy was unlikely. Among various peroxins responsible for peroxisome biogenesis, Pex19p functions as a chaperone protein for the transportation of peroxisomal membrane proteins. Immunoprecipitation analysis showed that PLA/AT-3 binds to Pex19p through its N-terminal proline-rich and C-terminal hydrophobic domains. The protein level and enzyme activity of PLA/AT-3 were increased by its coexpression with Pex19p. Moreover, PLA/AT-3 inhibited the binding of Pex19 to peroxisomal membrane proteins, such as Pex3p and Pex11ßp. A catalytically inactive point mutant of PLA/AT-3 could bind to Pex19p but did not inhibit the chaperone activity of Pex19p. Altogether, these results suggest a novel regulatory mechanism for peroxisome biogenesis through the interaction between Pex19p and PLA/AT-3.


Assuntos
Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Fosfolipases A2 Independentes de Cálcio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células COS , Cercopithecus aethiops , Regulação para Baixo , Células HEK293 , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Biológicos , Peroxinas , Fosfolipases A2 Independentes de Cálcio/química , Fosfolipases A2 Independentes de Cálcio/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
19.
J Lipid Res ; 56(3): 644-52, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25601960

RESUMO

ABCB4, which is specifically expressed on the canalicular membrane of hepatocytes, exports phosphatidylcholine (PC) into bile. Because SM depletion increases cellular PC content and stimulates PC and cholesterol efflux by ABCA1, a key transporter involved in generation of HDL, we predicted that SM depletion also stimulates PC efflux through ABCB4. To test this prediction, we compared the lipid efflux activity of ABCB4 and ABCA1 under SM depletion induced by two different types of inhibitors for SM synthesis, myriocin and (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide, in human embryonic kidney 293 and baby hamster kidney cells. Unexpectedly, SM depletion exerted opposite effects on ABCB4 and ABCA1, suppressing PC efflux through ABCB4 while stimulating efflux through ABCA1. Both ABCB4 and ABCA1 were recovered from Triton-X-100-soluble membranes, but ABCB4 was mainly recovered from CHAPS-insoluble SM-rich membranes, whereas ABCA1 was recovered from CHAPS-soluble membranes. These results suggest that a SM-rich membrane environment is required for ABCB4 to function. ABCB4 must have evolved to exert its maximum activity in the SM-rich membrane environment of the canalicular membrane, where it transports PC as the physiological substrate.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Fosfatidilcolinas/metabolismo , Esfingomielinas/metabolismo , Transportador 1 de Cassete de Ligação de ATP/antagonistas & inibidores , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico Ativo/fisiologia , Membrana Celular/genética , Cricetinae , Células HEK293 , Humanos , Fosfatidilcolinas/genética , Esfingomielinas/genética
20.
Traffic ; 16(1): 19-34, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25262571

RESUMO

Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E.


Assuntos
Transporte Biológico/fisiologia , Homeostase/fisiologia , Retinoides/metabolismo , Proteínas Celulares de Ligação ao Retinol/metabolismo , Vitaminas/metabolismo , Animais , Humanos , Fosfatidilinositóis/metabolismo
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