RESUMO
In eukaryotic cells, chromosomes are confined to the nucleus, which is compartmentalized by the nuclear membranes; these are continuous with the endoplasmic reticulum membranes. Maintaining the homeostasis of these membranes is an important cellular activity performed by lipid metabolic enzymes. However, how lipid metabolic enzymes affect nuclear membrane functions remains to be elucidated. We found that the very-long-chain fatty acid elongase Elo2 is located in the nuclear membrane and prevents lethal defects associated with nuclear membrane ruptures in mutants of the nuclear membrane proteins Lem2 and Bqt4 in the fission yeast Schizosaccharomyces pombe. Lipid composition analysis shows that t20:0/24:0 phytoceramide (a conjugate of C20:0 phytosphingosine and C24:0 fatty acid) is a major ceramide species in S. pombe The quantity of this ceramide is reduced in the absence of Lem2, and restored by increased expression of Elo2. Furthermore, loss of S. pombe Elo2 can be rescued by its human orthologs. These results suggest that the conserved very-long-chain fatty acid elongase producing the ceramide component is essential for nuclear membrane integrity and cell viability in eukaryotes.This article has an associated First Person interview with the first author of the paper.
Assuntos
Acetiltransferases/metabolismo , Elongases de Ácidos Graxos/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Sphingolipids are multifunctional lipids. Among the sphingolipid-component sphingoid bases, 4,14-sphingadiene (SPD) is unique such that it has a cis double bond with a bent structure. Although SPD was discovered half a century ago, its tissue distribution, biosynthesis, and degradation remain poorly understood. Here, we established a specific and quantitative method for SPD measurement and found that SPD exists in a wide range of mammalian tissues. SPD was especially abundant in kidney, where the amount of SPD was ~2/3 of sphingosine, the most abundant sphingoid base in mammals. Although SPD is metabolized to ceramides and SPD 1-phosphate with almost the same efficiency as sphingosine, it is less susceptible to degradation by a cleavage reaction, at least in vitro. We identified the fatty acid desaturase family protein FADS3 as a ceramide desaturase that produces SPD ceramides by desaturating ceramides containing sphingosine. SPD sphingolipids were preferentially localized outside lipid microdomains, suggesting that SPD has different functions compared to other sphingoid bases in the formation of lipid microdomains. In summary, we revealed the biosynthesis and degradation pathways of SPD and its characteristic membrane localization. Our findings contribute to the elucidation of the molecular mechanism underlying the generation of sphingolipid diversity.
Assuntos
Ceramidas/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Esfingosina/metabolismo , Animais , Ácidos Graxos Dessaturases/genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Esfingosina/análogos & derivadosRESUMO
Insulation by myelin lipids is essential to fast action potential conductivity: changes in their quality or amount can cause several neurologic disorders. Sjögren-Larsson syndrome (SLS) is one such disorder, which is caused by mutations in the fatty aldehyde dehydrogenase ALDH3A2. To date, the molecular mechanism underlying SLS pathology has remained unknown. In this study, we found that Aldh3a2 is expressed in oligodendrocytes and neurons in the mouse brain, and neurons of Aldh3a2 knockout (KO) mice exhibited impaired metabolism of the long-chain base, a component of sphingolipids. Aldh3a2 KO mice showed several abnormalities corresponding to SLS symptoms in behavioral tests, including increased paw slips on a balance beam and light-induced anxiety. In their brain tissue, 2-hydroxygalactosylceramide, an important lipid for myelin function and maintenance, was reduced by the inactivation of fatty acid 2-hydroxylase. Our findings provide important new insights into the molecular mechanisms responsible for neural pathogenesis caused by lipid metabolism abnormalities.-Kanetake, T., Sassa, T., Nojiri, K., Sawai, M., Hattori, S., Miyakawa, T., Kitamura, T., Kihara, A. Neural symptoms in a gene knockout mouse model of Sjögren-Larsson syndrome are associated with a decrease in 2-hydroxygalactosylceramide.
Assuntos
Comportamento Animal , Galactosilceramidas/deficiência , Síndrome de Sjogren-Larsson/fisiopatologia , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Ansiedade/metabolismo , Depressão/metabolismo , Galactosilceramidas/genética , Humanos , Luz , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Síndrome de Sjogren-Larsson/genética , Síndrome de Sjogren-Larsson/metabolismoRESUMO
Differences among fatty acids (FAs) in chain length and number of double bonds create lipid diversity. FA elongation proceeds via a four-step reaction cycle, in which the 3-hydroxyacyl-CoA dehydratases (HACDs) HACD1-4 catalyze the third step. However, the contribution of each HACD to 3-hydroxyacyl-CoA dehydratase activity in certain tissues or in different FA elongation pathways remains unclear. HACD1 is specifically expressed in muscles and is a myopathy-causative gene. Here, we generated Hacd1 KO mice and observed that these mice had reduced body and skeletal muscle weights. In skeletal muscle, HACD1 mRNA expression was by far the highest among the HACDs However, we observed only an â¼40% reduction in HACD activity and no changes in membrane lipid composition in Hacd1-KO skeletal muscle, suggesting that some HACD activities are redundant. Moreover, when expressed in yeast, both HACD1 and HACD2 participated in saturated and monounsaturated FA elongation pathways. Disruption of HACD2 in the haploid human cell line HAP1 significantly reduced FA elongation activities toward both saturated and unsaturated FAs, and HACD1 HACD2 double disruption resulted in a further reduction. Overexpressed HACD3 exhibited weak activity in saturated and monounsaturated FA elongation pathways, and no activity was detected for HACD4. We therefore conclude that HACD1 and HACD2 exhibit redundant activities in a wide range of FA elongation pathways, including those for saturated to polyunsaturated FAs, with HACD2 being the major 3-hydroxyacyl-CoA dehydratase. Our findings are important for furthering the understanding of the molecular mechanisms in FA elongation and diversity.
Assuntos
Ácidos Graxos/metabolismo , Hidroliases/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/enzimologia , Mioblastos Esqueléticos/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Animais , Sistemas CRISPR-Cas , Domínio Catalítico , Linhagem Celular Tumoral , Células Cultivadas , Ácidos Graxos/química , Regulação Enzimológica da Expressão Gênica , Humanos , Hidroliases/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos Knockout , Estrutura Molecular , Peso Molecular , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Doenças Musculares/enzimologia , Doenças Musculares/genética , Doenças Musculares/patologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/patologia , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
Although most mammalian fatty acids (FAs) are straight-chain, there also exist branched-chain FAs such as iso- and anteiso-FAs, especially in the meibomian glands. Meibum lipids, which are secreted from the meibomian glands and are important for dry eye prevention, contain abundant branched-chain lipids, such as cholesteryl esters and wax esters with chain-lengths of ≥C21 (very-long-chain; VLC). However, the exact tissue distribution of branched-chain lipids or the enzymes involved in the production of branched-chain VLCFAs has remained poorly understood. Here, we revealed that FA elongases ELOVL1, ELOVL3, and ELOVL7, of the seven mammalian ELOVL isozymes, elongated saturated branched-chain acyl-CoAs. ELOVL3 was highly active toward iso-C17:0 and anteiso-C17:0 acyl-CoAs and elongated them up to iso-C23:0 and anteiso-C25:0 acyl-CoAs, respectively. ELOVL1 elongated both iso- and anteiso-C23:0 acyl-CoAs to C25:0 acyl-CoAs. By establishing a liquid chromatography-tandem mass spectrometry method capable of separating branched- and straight-chain lipids, we showed that essentially all of the cholesteryl esters and 88% of the wax esters in the mouse meibomian glands are branched. In Elovl1 mutant mice, the levels of ≥C24:0 branched-chain cholesteryl esters and ≥C25:0 branched-chain wax esters were decreased, indicating that ELOVL1 indeed elongates branched-chain VLC acyl-CoAs in vivo. In addition, substantial amounts of ceramides containing branched-chain FAs were present in the skin, meibomian glands, and liver. Our findings provide new insights into the molecular mechanisms that create FA and lipid diversity.
Assuntos
Acil Coenzima A/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Elongases de Ácidos Graxos/metabolismo , Glândulas Tarsais/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Aminoácidos de Cadeia Ramificada/classificação , Animais , Ceramidas/classificação , Ceramidas/metabolismo , Ésteres do Colesterol/classificação , Ésteres do Colesterol/metabolismo , Elongases de Ácidos Graxos/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lipidômica/métodos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/metabolismo , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismo , Especificidade por SubstratoRESUMO
[This corrects the article DOI: 10.1016/j.isci.2021.102478.].
RESUMO
A lipid layer consisting of meibum lipids exists in the tear film and functions in preventing dry eye disease. Although the meibum lipids include diverse lipid classes, the synthesis pathway and role of each class remain largely unknown. Here, we created single and double knockout (KO and DKO, respectively) mice for the two acyl-CoA wax alcohol acyltransferases (Awat1 and Awat2) and investigated their dry eye phenotypes and meibum lipid composition. Awat2 KO and DKO mice exhibited severe dry eye with meibomian gland dysfunction, whereas Awat1 KO mice had mild dry eye. In these mice, specific meibum lipid classes were reduced: (O-acyl)-ω-hydroxy fatty acids and type 1ω wax diesters in Awat1 KO mice, wax monoesters and types 1ω and 2ω wax diesters in Awat2 KO mice, and most of these in DKO mice. Our findings reveal that Awat1 and Awat2 show characteristic substrate specificity and together produce diverse meibum lipids.
RESUMO
Meibum lipids form a lipid layer on the outermost side of the tear film and function to prevent water evaporation and reduce surface tension. (O-Acyl)-ω-hydroxy fatty acids (OAHFAs), a subclass of these lipids, are thought to be involved in connecting the lipid and aqueous layers in tears, although their actual function and synthesis pathway have to date remained unclear. Here, we reveal that the fatty acid ω-hydroxylase Cyp4f39 is involved in OAHFA production. Cyp4f39-deficient mice exhibited damaged corneal epithelium and shortening of tear film break-up time, both indicative of dry eye disease. In addition, tears accumulated on the lower eyelid side, indicating increased tear surface tension. In Cyp4f39-deficient mice, the production of wax diesters (type 1ω and 2ω) and cholesteryl OAHFAs was also impaired. These OAHFA derivatives show intermediate polarity among meibum lipids, suggesting that OAHFAs and their derivatives contribute to lipid polarity gradient formation for tear film stabilization.
Assuntos
Família 4 do Citocromo P450/fisiologia , Síndromes do Olho Seco/prevenção & controle , Ácidos Graxos/química , Lágrimas/química , Animais , Família 4 do Citocromo P450/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The skin permeability barrier is indispensable for maintaining water inside the body and preventing the invasion of pathogens and allergens; abnormalities lead to skin disorders such as atopic dermatitis and ichthyosis. Acylceramide is an essential lipid for skin barrier formation, and CYP4F22 is a fatty acid ω-hydroxylase involved in its synthesis. Mutations in CYP4F22 cause autosomal recessive congenital ichthyosis, although the symptoms vary among mutation sites and types. Here, we generated knockout mice deficient in Cyp4f39, the mouse ortholog of human CYP4F22, to investigate the effects of completely abrogating the function of the fatty acid ω-hydroxylase involved in acylceramide production on skin barrier formation. Cyp4f39 knockout mice died within 8 hours of birth. Large increases in transepidermal water loss and penetration of a dye from outside the body were observed, indicating severe skin barrier dysfunction. Histologic analyses of the epidermis revealed impairment of lipid lamella formation, accumulation of corneodesmosomes in the stratum corneum, and persistence of periderm. In addition, lipid analyses by mass spectrometry showed almost complete loss of acylceramide and its precursor ω-hydroxy ceramide. In conclusion, our findings provide clues to the molecular mechanisms of skin barrier abnormalities and the pathogenesis of ichthyosis caused by Cyp4f39 and CYP4F22 by association.
Assuntos
Ceramidas/biossíntese , Família 4 do Citocromo P450/metabolismo , Células Epidérmicas/patologia , Epiderme/patologia , Ictiose/patologia , Animais , Família 4 do Citocromo P450/genética , Desmossomos/patologia , Modelos Animais de Doenças , Células Epidérmicas/citologia , Feminino , Humanos , Ictiose/diagnóstico , Ictiose/genética , Masculino , Camundongos , Camundongos Knockout , Permeabilidade , Índice de Gravidade de DoençaRESUMO
Sphingolipids are multifunctional lipids and a major constituent of the cell membranes of eukaryotes. Although the fatty acid (FA) moiety of sphingolipids is usually a saturated or monounsaturated FA, polyunsaturated FA (PUFA)-containing species also exist in mammalian tissues. In the present study, we showed that C24:2 PUFA-containing ceramide is one of the seven major ceramide species in a wide range of tissues. C24:2 ceramide levels were especially high in spleen and small intestine; in the former, it was the fourth most abundant ceramide species. However, both the synthetic pathway and the physiological function of C24:2 ceramide had yet to be identified. Tracer analysis using deuterium-labeled linoleic acid (C18:2) revealed that C24:2 ceramide is produced via elongation of linoleic acid. We also found that the FA elongase ELOVL1 and the ceramide synthase CERS2 were involved in C24:2 ceramide production. Sphingolipids are known to form lipid microdomains in membranes; however, in a detergent-resistant membrane (DRM) assay, we observed a lower proportion of C24:2 sphingomyelin in the DRM fraction than of saturated sphingomyelins, suggesting that C24:2 sphingolipids may act to negatively regulate lipid microdomain formation. Our findings expand our knowledge of sphingolipid diversity, and provide insight into how different sphingolipid molecular species play different functions in biological membranes.