RESUMO
Structural genomic variations represent a major driving force of evolution, and a burst of large segmental gene duplications occurred in the human lineage during its separation from nonhuman primates. SRGAP2, a gene recently implicated in neocortical development, has undergone two human-specific duplications. Here, we find that both duplications (SRGAP2B and SRGAP2C) are partial and encode a truncated F-BAR domain. SRGAP2C is expressed in the developing and adult human brain and dimerizes with ancestral SRGAP2 to inhibit its function. In the mouse neocortex, SRGAP2 promotes spine maturation and limits spine density. Expression of SRGAP2C phenocopies SRGAP2 deficiency. It underlies sustained radial migration and leads to the emergence of human-specific features, including neoteny during spine maturation and increased density of longer spines. These results suggest that inhibition of SRGAP2 function by its human-specific paralogs has contributed to the evolution of the human neocortex and plays an important role during human brain development.
Assuntos
Encéfalo/citologia , Encéfalo/embriologia , Proteínas Ativadoras de GTPase/genética , Duplicação Gênica , Neurônios/citologia , Duplicações Segmentares Genômicas , Animais , Movimento Celular , Espinhas Dendríticas/metabolismo , Evolução Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Estrutura Terciária de Proteína , Especificidade da EspécieRESUMO
During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in nonneuronal cells filopodia dynamics decrease the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results suggest that F-BAR domains are functionally diverse and highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Neurogênese , Neurônios/citologia , Animais , Movimento Celular , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Proteínas Ativadoras de GTPase , Camundongos , Pseudópodes/metabolismoRESUMO
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
BACKGROUND: Very long-chain fatty acids (VLCFAs) are essential for functioning of biological membranes. ELOVL fatty acid elongase 1 catalyses elongation of saturated and monounsaturated C22-C26-VLCFAs. We studied two patients with a dominant ELOVL1 mutation. Independently, Kutkowska-Kazmierczak et al. had investigated the same patients and found the same mutation. We extended our study towards additional biochemical, functional, and therapeutic aspects. METHODS: We did mutation screening by whole exome sequencing. RNA-sequencing was performed in patient and control fibroblasts. Ceramide and sphingomyelin levels were measured by LC-MS/MS. ELOVL1 activity was determined by a stable isotope-labelled [13C]malonyl-CoA elongation assay. ELOVL1 expression patterns were investigated by immunofluorescence, in situ hybridisation and RT-qPCR. As treatment option, we investigated VLCFA loading of fibroblasts. RESULTS: Both patients carried an identical heterozygous de novo ELOVL1 mutation (c.494C>T, NM_001256399; p.S165F) not deriving from a founder allele. Patients suffered from epidermal hyperproliferation and increased keratinisation (ichthyosis). Hypomyelination of the central white matter explained spastic paraplegia and central nystagmus, while optic atrophy was causative for reduction of peripheral vision and visual acuity. The mutation abrogated ELOVL1 enzymatic activity and reduced ≥C24 ceramides and sphingomyelins in patient cells. Fibroblast loading with C22:0-VLCFAs increased C24:0-ceramides and sphingomyelins. We found competitive inhibition for ceramide and sphingomyelin synthesis between saturated and monounsaturated VLCFAs. Transcriptome analysis revealed upregulation of modules involved in epidermal development and keratinisation, and downregulation of genes for neurodevelopment, myelination, and synaptogenesis. Many regulated genes carried consensus proliferator-activated receptor (PPAR)α and PPARγ binding motifs in their 5'-regions. CONCLUSION: A dominant ELOVL1 mutation causes a neuro-ichthyotic disorder possibly amenable to treatment with PPAR-modulating drugs.
Assuntos
Acantose Nigricans/genética , Surdez/genética , Doenças Desmielinizantes/genética , Elongases de Ácidos Graxos/genética , Ictiose/genética , Mutação , Atrofia Óptica/genética , Paraplegia/genética , Acantose Nigricans/diagnóstico , Adolescente , Sequência de Aminoácidos , Biomarcadores , Biópsia , Pré-Escolar , Surdez/diagnóstico , Doenças Desmielinizantes/diagnóstico , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Genótipo , Humanos , Ictiose/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Atrofia Óptica/diagnóstico , Paraplegia/diagnóstico , Linhagem , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fenótipo , Sequenciamento do ExomaRESUMO
Lipids secreted from the meibomian gland (meibum) form the superficial layer of the tear film and prevent water evaporation from the ocular surface and infection. Here, we identified the fatty acid (FA) elongases responsible for the synthesis of very long-chain FAs (VLCFAs) that constitute the meibum lipids. Elongation of VLCFAs (ELOVL)1 is primarily responsible for the production of saturated VLCFAs, whereas ELOVL1, ELOVL3, and ELOVL4 redundantly participate in the synthesis of monounsaturated VLCFAs. Gene disruption of Elovl1 in mice shortened acyl moieties in the 2 major meibum lipids: cholesteryl esters and wax esters. These changes were associated with dry eye phenotypes, including increases in eye-blink frequency and water evaporation from the ocular surface at younger ages. Aged Elovl1 mutant mice developed corneal opacity with vascular invasion, accompanied by epidermalization of the cornea. Thus, in addition to the well-known VLC ceramides (acylceramides) in the epidermis, VLC meibum lipids are barrier-forming lipids.-Sassa, T., Tadaki, M., Kiyonari, H., Kihara, A. Very long-chain tear film lipids produced by fatty acid elongase ELOVL1 prevent dry eye disease in mice.
Assuntos
Acetiltransferases/metabolismo , Síndromes do Olho Seco/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Lágrimas/metabolismo , Acetiltransferases/genética , Animais , Opacidade da Córnea/genética , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Síndromes do Olho Seco/genética , Síndromes do Olho Seco/patologia , Síndromes do Olho Seco/prevenção & controle , Elongases de Ácidos Graxos , Camundongos , Camundongos TransgênicosRESUMO
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
Ceramide and complex sphingolipids regulate important cellular functions including cell growth, apoptosis, and signaling. Dysregulation of sphingolipid metabolism leads to pathological consequences such as sphingolipidoses and insulin resistance. Ceramides in mammals vary greatly in their acyl-chain composition: six different ceramide synthase isozymes (CERS1-6) that exhibit distinct substrate specificity and tissue distribution account for this diversity. In the present study, we demonstrated that CERS2-6 were phosphorylated at the cytoplasmic C-terminal regions. Most of the phosphorylated residues conformed to a consensus motif for phosphorylation by casein kinase 2 (CK2), and treatment of cells with the CK2-specific inhibitor CX-4945 lowered the phosphorylation levels of CERS2, -4, -5, and -6. Phosphorylation of CERS2 was especially important for its catalytic activity, acting mainly by increasing itsVmaxvalue. Phosphorylation modestly increased the catalytic activities of CERS4 and -5 and mildly increased those of CERS3 and -6. Dephosphorylation of endogenous ceramide synthases in the mouse brain led to severely reduced activity toward the Cers2 substrates C22:0/C24:0-CoAs and modestly reduced activity toward the Cers5/6 substrate C16:0-CoA. These results suggest that the phosphorylation of ceramide synthases may be a key regulatory point in the control of the distribution and levels of sphingolipids of various acyl-chain lengths.
Assuntos
Encéfalo/metabolismo , Caseína Quinase II/metabolismo , Esfingolipídeos/biossíntese , Esfingosina N-Aciltransferase/metabolismo , Animais , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Células HEK293 , Humanos , Camundongos , Naftiridinas/farmacologia , Fenazinas , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Esfingolipídeos/genética , Esfingosina N-Aciltransferase/genética , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologiaRESUMO
The fatty aldehyde dehydrogenase (FALDH) ALDH3A2 is the causative gene of Sjögren Larsson syndrome (SLS). To date, the molecular mechanism underlying the symptoms characterizing SLS has been poorly understood. Using Aldh3a2(-/-) mice, we found here that Aldh3a2 was the major FALDH active in undifferentiated keratinocytes. Long-chain base metabolism was greatly impaired in Aldh3a2(-/-) keratinocytes. Phenotypically, the intercellular spaces were widened in the basal layer of the Aldh3a2(-/-) epidermis due to hyperproliferation of keratinocytes. Furthermore, oxidative stress-induced genes were up-regulated in Aldh3a2(-/-) keratinocytes. Upon keratinocyte differentiation, the activity of another FALDH, Aldh3b2, surpassed that of Aldh3a2 As a result, Aldh3a2(-/-) mice were indistinguishable from wild-type mice in terms of their whole epidermis FALDH activity, and their skin barrier function was uncompromised under normal conditions. However, perturbation of the stratum corneum caused increased transepidermal water loss and delayed barrier recovery in Aldh3a2(-/-) mice. In conclusion, Aldh3a2(-/-) mice replicated some aspects of SLS symptoms, especially at the basal layer of the epidermis. Our results suggest that hyperproliferation of keratinocytes via oxidative stress responses may partly contribute to the ichthyosis symptoms of SLS.
Assuntos
Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/fisiologia , Permeabilidade da Membrana Celular , Queratinócitos/citologia , Síndrome de Sjogren-Larsson/patologia , Pele/patologia , Aldeído Oxirredutases/genética , Animais , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Feminino , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Sjogren-Larsson/etiologia , Pele/metabolismoRESUMO
X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by impaired degradation of very long-chain fatty acids (VLCFAs) due to mutations in the ABCD1 gene responsible for VLCFA transport into peroxisomes. Lorenzo's oil, a 4:1 mixture of glyceryl trioleate and glyceryl trierucate, has been used to reduce the saturated VLCFA level in the plasma of X-ALD patients; however, the mechanism by which this occurs remains elusive. We report the biochemical characterization of Lorenzo's oil activity toward elongation of very long-chain fatty acid (ELOVL) 1, the primary enzyme responsible for the synthesis of saturated and monounsaturated VLCFAs. Oleic and erucic acids inhibited ELOVL1, and, moreover, their 4:1 mixture (the FA composition of Lorenzo's oil) exhibited the most potent inhibitory activity. The kinetics analysis revealed that this was a mixed (not a competitive) inhibition. At the cellular level, treatment with the 4:1 mixture reduced the level of SM with a saturated VLCFA accompanied by an increased level of SM with a monounsaturated VLCFA, probably due to the incorporation of erucic acid into the FA elongation cycle. These results suggest that inhibition of ELOVL1 may be an underlying mechanism by which Lorenzo's oil exerts its action.
Assuntos
Acetiltransferases/antagonistas & inibidores , Ácidos Erúcicos/farmacologia , Ácidos Graxos/metabolismo , Esfingomielinas/metabolismo , Trioleína/farmacologia , Acetiltransferases/genética , Acetiltransferases/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Elongases de Ácidos Graxos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Cinética , Ácido Oleico/farmacologia , Ácido Palmítico/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácidos Esteáricos/farmacologiaRESUMO
Very long-chain fatty acids (VLCFAs, chain length >C20) exist in tissues throughout the body and are synthesized by repetition of the fatty acid (FA) elongation cycle composed of four successive enzymatic reactions. In mammals, the TER gene is the only gene encoding trans-2-enoyl-CoA reductase, which catalyzes the fourth reaction in the FA elongation cycle. The TER P182L mutation is the pathogenic mutation for nonsyndromic mental retardation. This mutation substitutes a leucine for a proline residue at amino acid 182 in the TER enzyme. Currently, the mechanism by which the TER P182L mutation causes nonsyndromic mental retardation is unknown. To understand the effect of this mutation on the TER enzyme and VLCFA synthesis, we have biochemically characterized the TER P182L mutant enzyme using yeast and mammalian cells transfected with the TER P182L mutant gene and analyzed the FA elongation cycle in the B-lymphoblastoid cell line with the homozygous TER P182L mutation (TER(P182L/P182L) B-lymphoblastoid cell line). We have found that TER P182L mutant enzyme exhibits reduced trans-2-enoyl-CoA reductase activity and protein stability, thereby impairing VLCFA synthesis and, in turn, altering the sphingolipid profile (i.e. decreased level of C24 sphingomyelin and C24 ceramide) in the TER(P182L/P182L) B-lymphoblastoid cell line. We have also found that in addition to the TER enzyme-catalyzed fourth reaction, the third reaction in the FA elongation cycle is affected by the TER P182L mutation. These findings provide new insight into the biochemical defects associated with this genetic mutation.
Assuntos
Ceramidas/metabolismo , Ácidos Graxos/biossíntese , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação de Sentido Incorreto , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Esfingomielinas/metabolismo , Estabilidade Enzimática , Células HEK293 , Células HeLa , Homozigoto , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: The epidermis contains many structurally diverse ceramides, which form the skin permeability barrier (skin barrier). Mutations in genes involved in the synthesis of ω-O-acylceramides (acylceramides) and protein-bound ceramides cause ichthyosis. OBJECTIVE: We aimed to elucidate the relationship between the degree of skin barrier impairment and changes in epidermal ceramide profiles caused by mutations in acylceramide synthesis genes. METHODS: Knockout (KO) mice of three genes-fatty acid (FA) ω-hydroxylase Cyp4f39 (human CYP4F22 ortholog), FA elongase Elovl1, and acyl-CoA synthetase Fatp4-were subjected to transepidermal water loss measurement, toluidine blue staining, and epidermal ceramide profiling via liquid chromatography coupled with tandem mass spectrometry. RESULTS: Transepidermal water loss was highest in Cyp4f39 KO mice, followed by Elovl1 KO and Fatp4 KO mice, and Cyp4f39 KO mice also showed the strongest degree of toluidine blue staining. In Cyp4f39 KO, Elovl1 KO, and Fatp4 KO mice, acylceramide levels were 0.6%, 1.6%, and 12%, respectively, of those in wild-type mice. Protein-bound ceramide levels were 0.2%, 30%, and 33%, respectively, of those in wild-type mice. We also observed a near-complete absence of ω-hydroxy ceramides in Cyp4f39 KO mice, reduced total ceramide levels and shortened FA moieties in Elovl1 KO mice, and increased hydroxylated ceramide levels and slightly shortened FA moieties in Fatp4 KO mice. CONCLUSIONS: The degree of reduction in protein-bound ceramide levels is probably related to the severity of skin barrier defects in these three strains. However, reduced acylceramide levels and other changes in ceramide composition unique to each KO strain are also involved.
Assuntos
Ceramidas , Pele , Animais , Humanos , Camundongos , Epiderme , Camundongos Knockout , Cloreto de Tolônio , ÁguaRESUMO
Sphingolipids, major lipid components of the eukaryotic plasma membrane, have a variety of physiological functions and have been associated with many diseases. They have also been implicated in apoptosis. Sphingolipids are heterogeneous in their acyl chain length, with long-chain (C16) and very long-chain (C24) sphingolipids being predominant in most mammalian tissues. We demonstrate that knockdown of ELOVL1 or CERS2, which catalyze synthesis of C24 acyl-CoAs and C24 ceramide, respectively, drastically reduced C24 sphingolipid levels with a complementary increase in C16 sphingolipids. Under ELOVL1 or CERS2 knockdown conditions, cisplatin-induced apoptosis significantly increased. Enhanced sensitivity to cisplatin-induced apoptosis exhibited close correlation with increases in caspase-3/7 activity. No significant alterations in sphingolipid metabolism such as ceramide generation were apparent with the cisplatin-induced apoptosis, and inhibitors of ceramide generation had no effect on the apoptosis. Apoptosis induced by UV radiation or C6 ceramides also increased in ELOVL1 or CERS2 knockdown cells. Changes in the composition of sphingolipid chain length may affect susceptibility to stimuli-induced apoptosis by affecting the properties of cell membranes, such as lipid microdomain/raft formation.
Assuntos
Acetiltransferases/genética , Apoptose , Ceramidas/biossíntese , Microdomínios da Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Esfingosina N-Aciltransferase/genética , Proteínas Supressoras de Tumor/genética , Acetiltransferases/deficiência , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Ceramidas/agonistas , Ceramidas/antagonistas & inibidores , Ceramidas/farmacologia , Cisplatino/farmacologia , Colorimetria , Elongases de Ácidos Graxos , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Metabolismo dos Lipídeos , Microdomínios da Membrana/efeitos da radiação , Proteínas de Membrana/deficiência , RNA Interferente Pequeno/genética , Esfingosina N-Aciltransferase/deficiência , Transfecção , Proteínas Supressoras de Tumor/deficiência , Raios UltravioletaRESUMO
One of the most fascinating questions in evolutionary biology is how traits unique to humans, such as their high cognitive abilities, erect bipedalism, and hairless skin, are encoded in the genome. Recent advances in genomics have begun to reveal differences between the genomes of the great apes. It has become evident that one of the many mutation types, segmental duplication, has drastically increased in the primate genomes, and most remarkably in the human genome. Genes contained in these segmental duplications have a tremendous potential to cause genetic innovation, probably accounting for the acquisition of human-specific traits. In this review, I begin with an overview of the genes, which have increased their copy number specifically in the human lineage, following its separation from the common ancestor with our closest living relative, the chimpanzee. Then, I introduce the recent experimental approaches, focusing on SRGAP2, which has been partially duplicated, to elucidate the role of SRGAP2 protein and its human-specific paralogs in human brain development and evolution.
Assuntos
Evolução Biológica , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteínas Ativadoras de GTPase/genética , Duplicação Gênica , Animais , Proteínas Ativadoras de GTPase/metabolismo , Genoma Humano , Genômica , Humanos , Especificidade da EspécieRESUMO
Very long-chain fatty acids (VLCFAs) exert a variety of cellular functions and are associated with numerous diseases. However, the precise pathway behind their elongation has remained elusive. Moreover, few regulatory mechanisms for VLCFAs synthesis have been identified. Elongases catalyze the first of four steps in the VLCFA elongation cycle; mammals have seven elongases (ELOVL1-7). In the present study, we determined the precise substrate specificities of all the ELOVLs by in vitro analyses. Particularly notable was the high activity exhibited by ELOVL1 toward saturated and monounsaturated C20- and C22-CoAs, and that it was essential for the production of C24 sphingolipids, which are unique in their capacity to interdigitate within the membrane as a result of their long chain length. We further established that ELOVL1 activity is regulated with the ceramide synthase CERS2, an enzyme essential for C24 sphingolipid synthesis. This regulation may ensure that the production of C24-CoA by elongation is coordinated with its utilization. Finally, knockdown of ELOVL1 caused a reduction in the activity of the Src kinase LYN, confirming that C24-sphingolipids are particularly important in membrane microdomain function.
Assuntos
Acetiltransferases/metabolismo , Acil Coenzima A/biossíntese , Ácidos Graxos/biossíntese , Esfingolipídeos/biossíntese , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Acil Coenzima A/química , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Elongases de Ácidos Graxos , Ácidos Graxos/química , Feminino , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Técnicas In Vitro , Masculino , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/química , Esfingosina N-Aciltransferase/metabolismo , Especificidade por Substrato , Distribuição Tecidual , Proteínas Supressoras de Tumor/metabolismoRESUMO
The permeability barrier present in the oral cavity is critical for protection from infection. Although lipids have properties suitable for permeability barrier formation, little is known about their role in oral barrier formation. Here, we show the presence of ω-O-acylceramides (acylceramides) and protein-bound ceramides, which are essential for the formation of permeability barriers in the epidermis, in the oral mucosae (buccal and tongue mucosae), esophagus, and stomach in mice. Conditional knockout of the fatty acid elongase Elovl1, which is involved in the synthesis of ≥C24 ceramides including acylceramides and protein-bound ceramides, in the oral mucosae and esophagus causes increased pigment penetration into the mucosal epithelium of the tongue and enhanced aversive responses to capsaicin-containing water. We find acylceramides in the buccal and gingival mucosae and protein-bound ceramides in the gingival mucosa in humans. These results indicate that acylceramides and protein-bound ceramides are important for oral permeability barrier formation.
Assuntos
Ceramidas , Epiderme , Humanos , Camundongos , Animais , Células Epidérmicas , Permeabilidade , GengivaRESUMO
Spinocerebellar ataxias (SCAs) are autosomal dominant diseases characterized by cerebellar atrophy and ataxia. The SCA subtype SCA34 is caused by specific mutations in the gene ELOVL4, which encodes a fatty acid (FA) elongase that synthesizes ultra-long-chain (ULC; ≥C26) FAs. However, the pathogenesis and molecular mechanism that confers dominant inheritance remains unknown. Here, a cell-based assay demonstrated that each of the five known SCA34 mutants produced shorter ULC polyunsaturated FA-containing phosphatidylcholines (ULC-PCs) than wild-type protein, in the following order of severity: Q180P and T233M > W246G > I171T and L168F. Next, we generated knock-in mouse embryonic stem cells that contained heterozygous Q180P, heterozygous W246G, or homozygous W246G mutations. Neuronal differentiation-dependent production of ULC-PCs was reduced in heterozygous Q180P and homozygous W246G cells relative to control cells, and we observed shortening of the FA moiety in all mutant cells. This FA shortening was consistent with our prediction that amino acid residues substituted by SCA34 mutations are located in the transmembrane helices that interact with the ω-end region of the FA moiety of the substrate acyl-CoA. Hence, reduced levels and shortening of ULC-PCs in neurons may cause SCA34, and incomplete elongation of ULC polyunsaturated acyl-CoAs by mutated ELOVL4 may induce dominant inheritance.
RESUMO
BACKGROUND: Sjögren-Larsson syndrome (SLS) is a neurocutaneous disorder whose causative gene is the fatty aldehyde dehydrogenase ALDH3A2 and of which ichthyosis is the major skin symptom. The stratum corneum contains a variety of ceramides, among which ω-O-acylceramides (acylceramides) and protein-bound ceramides are essential for skin permeability barrier formation. OBJECTIVES: To determine the ceramide classes/species responsible for SLS pathogenesis and the enzymes that are impaired in SLS. METHODS: Genomic DNA was collected from peripheral blood samples from an SLS patient and her parents, and whole-genome sequencing and Sanger sequencing were performed. Lipids were extracted from stratum corneum samples from the SLS patient and healthy volunteers and subjected to ceramide profiling via liquid chromatography coupled with tandem mass spectrometry. RESULTS: A duplication (c.55_130dup) and a missense mutation (p.Lys447Glu) were found in the patient's ALDH3A2 gene. The patient had reduced levels of all acylceramide classes, with total acylceramide levels at 25 % of healthy controls. Reductions were also observed for several nonacylated ceramides: ceramides with phytosphingosine or 6-hydroxysphingosine in the long-chain base moiety were reduced to 24 % and 41 % of control levels, respectively, and ceramides with an α-hydroxy fatty acid as the fatty acid moiety were reduced to 29 %. The fatty acid moiety was shortened in many nonacylated ceramide classes. CONCLUSION: These results suggest that reduced acylceramide levels are a primary cause of the ichthyosis symptoms of SLS, but reductions in other ceramide classes may also be involved.
Assuntos
Ictiose Lamelar , Ictiose , Síndrome de Sjogren-Larsson , Ceramidas/análise , Epiderme/patologia , Ácidos Graxos , Feminino , Humanos , Ictiose/genética , Ictiose/patologia , Ictiose Lamelar/patologia , Síndrome de Sjogren-Larsson/genética , Síndrome de Sjogren-Larsson/patologiaRESUMO
INTRODUCTION: Next generation sequencing technologies allow detection of very rare pathogenic gene variants and uncover cerebral palsy. Herein, we describe two siblings with cerebral palsy due to ELOVL1 splice site mutation in autosomal recessive manner. ELOVL1 catalyzes fatty acid elongation to produce very long-chain fatty acids (VLCFAs; ≥C21), most of which are components of sphingolipids such as ceramides and sphingomyelins. Ichthyotic keratoderma, spasticity, hypomyelination, and dysmorphic facies (MIM: 618527) stem from ELOVL1 gene deficiency in human. METHODS: We have studied a consanguineous family with whole exome sequencing (WES) and performed in depth analysis of cryptic splicing on the molecular level using RNA. Comprehensive analysis of ceramides in the skin stratum corneum of patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS). ELOVL1 protein structure was computationally modelled. RESULTS: The novel c.376-2A > G (ENST00000372458.8) homozygous variant in the affected siblings causes exon skipping. Comprehensive analysis of ceramides in the skin stratum corneum of patients using LC-MS/MS demonstrated significant shortening of fatty acid moieties and severe reduction in the levels of acylceramides. DISCUSSION: It has recently been shown that disease associated variants of ELOVL1 segregate in an autosomal dominant manner. However, our study for the first time demonstrates an alternative autosomal recessive inheritance model for ELOVL1. In conclusion, we suggest that in ultra-rare diseases, being able to identify the inheritance patterns of the disease-associated gene or genes can be an important guide to identifying the molecular mechanism of genetic cerebral palsy.
Assuntos
Paralisia Cerebral , Discinesias , Ictiose , Ceramidas/metabolismo , Paralisia Cerebral/genética , Cromatografia Líquida , Discinesias/genética , Éxons , Elongases de Ácidos Graxos , Ácidos Graxos , Humanos , Ictiose/genética , Imidazóis , Espasticidade Muscular/genética , Mutação/genética , Linhagem , Sulfonamidas , Espectrometria de Massas em Tandem , TiofenosRESUMO
Recent studies have demonstrated that the topography of thalamocortical (TC) axon projections is initiated before they reach the cortex, in the ventral telencephalon (VTel). However, at this point, the molecular mechanisms patterning the topography of TC projections in the VTel remains poorly understood. Here, we show that a long-range, high-rostral to low-caudal gradient of Netrin-1 in the VTel is required in vivo for the topographic sorting of TC axons to distinct cortical domains. We demonstrate that Netrin-1 is a chemoattractant for rostral thalamic axons but functions as a chemorepulsive cue for caudal thalamic axons. In accordance with this model, DCC is expressed in a high-rostromedial to low-caudolateral gradient in the dorsal thalamus (DTh), whereas three Unc5 receptors (Unc5A-C) show graded expression in the reverse orientation. Finally, we show that DCC is required for the attraction of rostromedial thalamic axons to the Netrin-1-rich, anterior part of the VTel, whereas DCC and Unc5A/C receptors are required for the repulsion of caudolateral TC axons from the same Netrin-1-rich region of the VTel. Our results demonstrate that a long-range gradient of Netrin-1 acts as a counteracting force from ephrin-A5 to control the topography of TC projections before they enter the cortex.
Assuntos
Axônios/metabolismo , Regulação da Expressão Gênica , Fatores de Crescimento Neural/metabolismo , Telencéfalo/metabolismo , Tálamo/citologia , Tálamo/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Padronização Corporal , Comunicação Celular , Fatores Quimiotáticos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Crescimento Neural/genética , Receptores de Netrina , Netrina-1 , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Telencéfalo/embriologia , Proteínas Supressoras de Tumor/genéticaRESUMO
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.