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1.
Nat Commun ; 11(1): 3317, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620775

RESUMO

Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis.


Assuntos
Embrião não Mamífero/metabolismo , Mitose , Receptores de Peptídeos/metabolismo , Esfingolipídeos/metabolismo , Via de Sinalização Wnt , Sequência de Aminoácidos , Animais , Divisão Celular Assimétrica/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Lipoilação , Tubo Neural/citologia , Tubo Neural/embriologia , Tubo Neural/metabolismo , Receptores de Peptídeos/genética , Homologia de Sequência de Aminoácidos , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Fuso Acromático/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(27): 15591-15598, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576697

RESUMO

Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of l-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C14 to C18, forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso-branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso-methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins.


Assuntos
Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Camundongos , Serina C-Palmitoiltransferase/genética , Especificidade por Substrato
3.
Enzyme Microb Technol ; 137: 109515, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423667

RESUMO

Serine palmitoyltransferase (SPTase), the first enzyme of the sphingolipid biosynthesis pathway, produces 3-ketodihydrosphingosine by a Claisen-like condensation/decarboxylation reaction of l-Ser and palmitoyl-CoA (n-C16-CoA). Previous structural analysis of Sphingomonas paucimobilis SPTase (SpSPTase) revealed a dynamic active site loop (RPPATP; amino acids 378-383) in which R378 (underlined) forms a salt bridge with the carboxylic acid group of the PLP : l-Ser external aldimine. We hypothesized that this interaction might play a key role in acyl group substrate selectivity and therefore performed site-saturation mutagenesis at position 378 based on semi-rational design to expand tolerance for shorter acyl-CoA's. The resulting library was initially screened for the reaction between l-Ser and dodecanoyl-CoA (n-C12-CoA). The most interesting mutant (R378 K) was then purified and compared to wild-type SpSPTase against a panel of acyl-CoA's. These data showed that the R378 K substitution shifted the acyl group preference to shorter chain lengths, opening the possibility of using this and other engineered variants for biocatalytic C-C bond-forming reactions.


Assuntos
Acil Coenzima A/metabolismo , Engenharia Metabólica/métodos , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Sphingomonas/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Especificidade por Substrato
4.
Nat Commun ; 11(1): 2471, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424203

RESUMO

Gut microbes are linked to host metabolism, but specific mechanisms remain to be uncovered. Ceramides, a type of sphingolipid (SL), have been implicated in the development of a range of metabolic disorders from insulin resistance (IR) to hepatic steatosis. SLs are obtained from the diet and generated by de novo synthesis in mammalian tissues. Another potential, but unexplored, source of mammalian SLs is production by Bacteroidetes, the dominant phylum of the gut microbiome. Genomes of Bacteroides spp. and their relatives encode serine palmitoyltransfease (SPT), allowing them to produce SLs. Here, we explore the contribution of SL-production by gut Bacteroides to host SL homeostasis. In human cell culture, bacterial SLs are processed by host SL-metabolic pathways. In mouse models, Bacteroides-derived lipids transfer to host epithelial tissue and the hepatic portal vein. Administration of B. thetaiotaomicron to mice, but not an SPT-deficient strain, reduces de novo SL production and increases liver ceramides. These results indicate that gut-derived bacterial SLs affect host lipid metabolism.


Assuntos
Bacteroides/fisiologia , Ceramidas/metabolismo , Microbioma Gastrointestinal , Redes e Vias Metabólicas , Esfingolipídeos/metabolismo , Animais , Células CACO-2 , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Vida Livre de Germes , Humanos , Resistência à Insulina , Mucosa Intestinal/microbiologia , Fígado/metabolismo , Redes e Vias Metabólicas/genética , Camundongos , Mutação/genética , Serina C-Palmitoiltransferase/deficiência , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo
5.
Am J Respir Cell Mol Biol ; 62(6): 783-792, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32078788

RESUMO

Polymorphism at the 17q21 gene locus and wheezing responses to rhinovirus (RV) early in childhood conspire to increase the risk of developing asthma. However, the mechanisms mediating this gene-environment interaction remain unclear. In this study, we investigated the impact of one of the 17q21-encoded genes, ORMDL3 (orosomucoid-like 3), on RV replication in human epithelial cells. ORMDL3 knockdown inhibited RV-A16 replication in HeLa, BEAS-2B, A549, and NCI-H358 epithelial cell lines and primary nasal and bronchial epithelial cells. Inhibition varied by RV species, as both minor and major group RV-A subtypes RV-B52 and RV-C2 were inhibited but not RV-C15 or RV-C41. ORMDL3 siRNA did not affect expression of the major group RV-A receptor ICAM-1 or initial internalization of RV-A16. The two major outcomes of ORMDL3 activity, SPT (serine palmitoyl-CoA transferase) inhibition and endoplasmic reticulum (ER) stress induction, were further examined: silencing ORMDL3 decreased RV-induced ER stress and IFN-ß mRNA expression. However, pharmacologic induction of ER stress and concomitant increased IFN-ß inhibited RV-A16 replication. Conversely, blockade of ER stress with tauroursodeoxycholic acid augmented replication, pointing to an alternative mechanism for the effect of ORMDL3 knockdown on RV replication. In comparison, the SPT inhibitor myriocin increased RV-A16 but not RV-C15 replication and negated the inhibitory effect of ORMDL3 knockdown. Furthermore, lipidomics analysis revealed opposing regulation of specific sphingolipid species (downstream of SPT) by myriocin and ORMDL3 siRNA, correlating with the effect of these treatments on RV replication. Together, these data revealed a requirement for ORMDL3 in supporting RV replication in epithelial cells via SPT inhibition.


Assuntos
Células Epiteliais/virologia , Proteínas de Membrana/fisiologia , Rhinovirus/fisiologia , Replicação Viral , Células A549 , Asma/etiologia , Brônquios/citologia , Células Cultivadas , Cromossomos Humanos Par 17/genética , Estresse do Retículo Endoplasmático , Ácidos Graxos Monoinsaturados/farmacologia , Predisposição Genética para Doença , Genótipo , Células HeLa , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Mucosa Nasal/citologia , Infecções por Picornaviridae/complicações , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Rhinovirus/genética , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Replicação Viral/efeitos dos fármacos
6.
Artigo em Inglês | MEDLINE | ID: mdl-31988047

RESUMO

Curcumin, a hydrophobic polyphenol found in the rhizome of Curcuma longa, has been shown to reduce intracellular lipid accumulation in mouse models of lysosomal storage diseases such as Niemann-Pick type C. Exosomes are small extracellular vesicles secreted by cells in response to changes in intracellular ceramide composition. Curcumin can induce exosome/microvesicle release in cellular models of lipid deposition; however, the mechanism by which curcumin stimulates this release is unknown. In a model of lipid trafficking impairment in C6 glia cells, we show that curcumin stimulated ceramide synthesis by increasing the intracellular concentration of ceramide-dihydroceramide. Ceramide overload increased exosome/microvesicle secretion 10-fold, thereby reducing the concentration of lipids in the endolysosomal compartment. These effects were blocked by inhibitors of serine palmitoyltransferase (myriocin) and ceramide synthase (fumonisin B1). It is concluded that the decrease in intracellular lipid deposition induced by curcumin is mediated by increased ceramide synthesis and exosome/microvesicle release. This action may represent an additional health benefit of curcumin.


Assuntos
Micropartículas Derivadas de Células/efeitos dos fármacos , Ceramidas/biossíntese , Curcumina/farmacologia , Exossomos/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Fumonisinas/farmacologia , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neuroglia/patologia , Doença de Niemann-Pick Tipo C/dietoterapia , Doença de Niemann-Pick Tipo C/patologia , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Ratos , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/metabolismo
7.
Cell Physiol Biochem ; 54(1): 110-125, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31999897

RESUMO

BACKGROUND/AIMS: Cystic Fibrosis (CF) is an inherited disease associated with a variety of mutations affecting the CFTR gene. A deletion of phenylalanine 508 (F508) affects more than 70% of patients and results in unfolded proteins accumulation, originating a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol and lipids accumulation, impaired autophagy at the cellular level. Lung inflammation has been extensively related to the accumulation of the lipotoxin ceramide. We recently proved that inhibition of ceramide synthesis by Myriocin reduces inflammation and ameliorates the defence response against pathogens infection, which is downregulated in CF. Here, we aim at demonstrating the mechanisms of Myriocin therapeutic effects in Cystic Fibrosis broncho-epithelial cells. METHODS: The effect of Myriocin treatment, on F508-CFTR bronchial epithelial cell line IB3-1 cells, was studied by evaluating the expression of key proteins and genes involved in autophagy and lipid metabolism, by western blotting and real time PCR. Moreover, the amount of glycerol-phospholipids, triglycerides, and cholesterols, sphingomyelins and ceramides were measured in treated and untreated cells by LC-MS. Finally, Sptlc1 was transiently silenced and the effect on ceramide content, autophagy and transcriptional activities was evaluated as above mentioned. RESULTS: We demonstrate that Myriocin tightly regulates metabolic function and cell resilience to stress. Myriocin moves a transcriptional program that activates TFEB, major lipid metabolism and autophagy regulator, and FOXOs, central lipid metabolism and anti-inflammatory/anti-oxidant regulators. The activity of these transcriptional factors is associated with the induction of PPARs nuclear receptors activity, whose targets are genes involved in lipid transport compartmentalization and oxidation. Transient silencing of SPTCL1 recapitulates the effects induced by Myriocin. CONCLUSION: Cystic Fibrosis bronchial epithelia accumulate lipids, exacerbating inflammation. Myriocin administration: i) activates the transcriptions of genes involved in enhancing autophagy-mediated stress clearance; ii) reduces the content of several lipid species and, at the same time, iii) enhances mitochondrial lipid oxidation. Silencing the expression of Sptlc1 reproduces Myriocin induced autophagy and transcriptional activities, demonstrating that the inhibition of sphingolipid synthesis drives a transcriptional program aimed at addressing cell metabolism towards lipid oxidation and at exploiting autophagy mediated clearance of stress. We speculate that regulating sphingolipid de novo synthesis can relieve from chronic inflammation, improving energy supply and anti-oxidant responses, indicating an innovative therapeutic strategy for CF.


Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Esfingolipídeos/metabolismo , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular , Colesterol/análise , Cromatografia Líquida de Alta Pressão , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Espectrometria de Massas , PPAR gama/genética , PPAR gama/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/análise , Esfingomielinas/análise
8.
Hum Mol Genet ; 29(2): 189-201, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31628463

RESUMO

Metabolites are small intermediate products of cellular metabolism perturbed in a variety of complex disorders. Identifying genetic markers associated with metabolite concentrations could delineate disease-related metabolic pathways in humans. We tested genetic variants for associations with 136 metabolites in 1954 Chinese from Singapore. At a conservative genome-wide threshold (3.7 × 10-10), we detected 1899 variant-metabolite associations at 16 genetic loci. Three loci (ABCA7, A4GALT, GSTM2) represented novel associations with metabolites, with the strongest association observed between ABCA7 and d18:1/24:1 dihexosylceramide. Among 13 replicated loci, we identified six new variants independent of previously reported metabolite or lipid signals. We observed variant-metabolite associations at two loci (ABCA7, CHCHD2) that have been linked to neurodegenerative diseases. At SGPP1 and SPTLC3 loci, genetic variants showed preferential selectivity for sphingolipids with d16 (rather than d18) sphingosine backbone, including sphingosine-1-phosphate (S1P). Our results provide new genetic associations for metabolites and highlight the role of metabolites as intermediate modulators in disease metabolic pathways.


Assuntos
Doença de Alzheimer/genética , Grupo com Ancestrais do Continente Asiático/genética , Glicoesfingolipídeos/metabolismo , Doença de Parkinson/genética , Esfingolipídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Doença de Alzheimer/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , China , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Glicoesfingolipídeos/genética , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Lisofosfolipídeos/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Doença de Parkinson/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Serina/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/química , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Reprod Biol Endocrinol ; 17(1): 81, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31647034

RESUMO

BACKGROUND: Gestational diabetes mellitus (GDM) is associated with important factors that influence fetal development. Sphingolipids are known to be associated with the development of diabetes. Our objective was to examine ceramide, a key sphingolipid, hyperosmolarity, and apoptosis in placentas from GDM patients treated with insulin or diet. METHODS: Ceramide levels were assessed in placental tissues using immunohistochemistry. Immunoblot was performed to quantify serine palmitoyltransferase (SPT), the rate-limiting enzyme in ceramide biosynthesis, NFAT5, SMIT, AR, caspase 3 and the X-linked inhibitor of apoptosis. Trophoblast cells were treated with insulin or ceramide and assessments for mitochondrial respiration, caspase 3 and XIAP were also performed. RESULTS: Immunohistochemistry showed increased ceramides in the placental villous trophoblasts of the insulin-treated GDM patients. Nuclear SPT was upregulated only in the insulin-treated GDM placenta when compared to controls. Nuclear NFAT5 was also increased in the GDM placenta. Active caspase 3 was elevated in placentas from both insulin- and diet-treated GDM patients. Mitochondrial respiration was decreased in trophoblasts treated with ceramide. Active caspase was not changed while XIAP protein was increased in trophoblasts treated with ceramide. CONCLUSIONS: Our findings confirm the presence of ceramide in the human placenta of control and GDM patients. Furthermore, we conclude that ceramide is increased in the placental trophoblast during insulin treatment and that its upregulation correlates with elevated NFAT5, SMIT, increased apoptosis and decreased trophoblast mitochondrial respiration.


Assuntos
Ceramidas/metabolismo , Diabetes Gestacional/metabolismo , Mitocôndrias/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Ceramidas/farmacologia , Diabetes Gestacional/tratamento farmacológico , Dieta , Feminino , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Gravidez , Serina C-Palmitoiltransferase/metabolismo , Trofoblastos/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
10.
Int J Mol Sci ; 20(20)2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614447

RESUMO

Ceramide and sphingosine display a unique profile during brain development, indicating their critical role in myelinogenesis. Employing advanced technology such as gas chromatography-mass spectrometry, high performance liquid chromatography, and immunocytochemistry, along with cell culture and molecular biology, we have found an accumulation of sphingosine in brain tissues of patients with multiple sclerosis (MS) and in the spinal cord of rats induced with experimental autoimmune encephalomyelitis. The elevated sphingosine leads to oligodendrocyte death and fosters demyelination. Ceramide elevation by serine palmitoyltransferse (SPT) activation was the primary source of the sphingosine elevation as myriocin, an inhibitor of SPT, prevented sphingosine elevation and protected oligodendrocytes. Supporting this view, fingolimod, a drug used for MS therapy, reduced ceramide generation, thus offering partial protection to oligodendrocytes. Sphingolipid synthesis and degradation in normal development is regulated by a series of microRNAs (miRNAs), and hence, accumulation of sphingosine in MS may be prevented by employing miRNA technology. This review will discuss the current knowledge of ceramide and sphingosine metabolism (synthesis and breakdown), and how their biosynthesis can be regulated by miRNA, which can be used as a therapeutic approach for MS.


Assuntos
Ceramidas/biossíntese , MicroRNAs/genética , Esclerose Múltipla/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingosina/biossíntese , Animais , Encéfalo/metabolismo , Cloridrato de Fingolimode/farmacologia , Cloridrato de Fingolimode/uso terapêutico , Humanos , MicroRNAs/antagonistas & inibidores , Terapia de Alvo Molecular , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Bainha de Mielina/metabolismo , Ratos , Serina C-Palmitoiltransferase/antagonistas & inibidores
11.
Biochem Biophys Res Commun ; 520(1): 1-7, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31554600

RESUMO

Serine palmitoyltransferase long chain-1 (SPTLC1), which is the rate-limiting enzyme for sphingolipid biosynthesis, has been indicated to be essential for carcinoma cell survival and proliferation in recent, but its role in the regulation of renal cell carcinoma (RCC) remains unknown. In the present study, we found that SPTLC1 expression was significantly decreased in RCC tissues compared to non-tumor tissues, and low SPTLC1 expression was associated with poor overall survival of RCC patients. In addition, our results revealed that forced expression of SPTLC1 could significantly inhibit cell growth in vitro and in vivo via, at least in part, modulating Akt/FOXO1 signaling pathway, thus representing a novel role of SPTLC1 in the regulation of tumor growth in RCC for the first time.


Assuntos
Carcinoma de Células Renais/metabolismo , Proteína Forkhead Box O1/metabolismo , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Animais , Carcinoma de Células Renais/patologia , Proliferação de Células , Humanos , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Serina C-Palmitoiltransferase/biossíntese , Células Tumorais Cultivadas
12.
Neurochem Res ; 44(9): 2113-2122, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31372925

RESUMO

To investigate the role and mechanism of microRNA-124-3p (miR-124-3p) and serine palmitoyltransferase long chain base subunit 2 (SPTLC2) in neuronal apoptosis induced by mechanical injury. Transient transfection was used to modify the expression of miR-124-3p and SPTLC2. After transfection, neuronal apoptosis was evaluated in an in vitro injury model of primary neurons using TUNEL staining and western blot. The correlation between miR-124-3p and SPTLC2 was identified through a dual luciferase reporter assay in HEK293 cells. A rescue experiment in primary neurons was performed to further confirm the result. To explore the downstream mechanisms, co-immunoprecipitation was performed to identify proteins that interact with SPTLC2 in toll-like receptor 4 (TLR4) signalling pathway. Subsequently, the relative expression levels of TLR4 pathway molecules were measured by western blot. Our results showed that increased miR-124-3p can inhibit neuronal apoptosis, which is opposite to the effect of SPTLC2. In addition, miR-124-3p was proved to negatively regulate SPTLC2 expression and suppress the apoptosis-promoting effect of SPTLC2 via the TLR4 signalling pathway.


Assuntos
Apoptose/fisiologia , MicroRNAs/fisiologia , Neurônios/fisiologia , Serina C-Palmitoiltransferase/fisiologia , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Córtex Cerebral/fisiologia , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Traumatismos do Sistema Nervoso/fisiopatologia
13.
Nutrients ; 11(5)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137828

RESUMO

Sulforaphane (SFA), a naturally active isothiocyanate compound from cruciferous vegetables used in clinical trials for cancer treatment, was found to possess potency to alleviate insulin resistance. But its underlying molecular mechanisms are still incompletely understood. In this study, we assessed whether SFA could improve insulin sensitivity and glucose homeostasis both in vitro and in vivo by regulating ceramide production. The effects of SFA on glucose metabolism and expression levels of key proteins in the hepatic insulin signaling pathway were evaluated in insulin-resistant human hepatic carcinoma HepG2 cells. The results showed that SFA dose-dependently increased glucose uptake and intracellular glycogen content by regulating the insulin receptor substrate 1 (IRS-1)/protein kinase B (Akt) signaling pathway in insulin-resistant HepG2 cells. SFA also reduced ceramide contents and downregulated transcription of ceramide-related genes. In addition, knockdown of serine palmitoyltransferase 3 (SPTLC3) in HepG2 cells prevented ceramide accumulation and alleviated insulin resistance. Moreover, SFA treatment improved glucose tolerance and insulin sensitivity, inhibited SPTLC3 expression and hepatic ceramide production and reduced hepatic triglyceride content in vivo. We conclude that SFA recovers glucose homeostasis and improves insulin sensitivity by blocking ceramide biosynthesis through modulating SPTLC3, indicating that SFA may be a potential candidate for prevention and amelioration of hepatic insulin resistance via a ceramide-dependent mechanism.


Assuntos
Ceramidas/biossíntese , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Hepatócitos/efeitos dos fármacos , Resistência à Insulina , Isotiocianatos/farmacologia , Fígado/efeitos dos fármacos , Serina C-Palmitoiltransferase/antagonistas & inibidores , Animais , Glicogênio/metabolismo , Células Hep G2 , Hepatócitos/enzimologia , Humanos , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Fígado/enzimologia , Masculino , Camundongos Endogâmicos C57BL , Ácido Palmítico/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismo
14.
J Biol Chem ; 294(23): 9213-9224, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31053639

RESUMO

Chronic or excess glucocorticoid exposure causes lipid disorders such as hypertriglyceridemia and hepatic steatosis. Angptl4 (angiopoietin-like 4), a primary target gene of the glucocorticoid receptor in hepatocytes and adipocytes, is required for hypertriglyceridemia and hepatic steatosis induced by the synthetic glucocorticoid dexamethasone. Angptl4 has also been shown to be required for dexamethasone-induced hepatic ceramide production. Here, we further examined the role of ceramide-mediated signaling in hepatic dyslipidemia caused by chronic glucocorticoid exposure. Using a stable isotope-labeling technique, we found that dexamethasone treatment induced the rate of hepatic de novo lipogenesis and triglyceride synthesis. These dexamethasone responses were compromised in Angptl4-null mice (Angptl4-/-). Treating mice with myriocin, an inhibitor of the rate-controlling enzyme of de novo ceramide synthesis, serine palmitoyltransferase long-chain base subunit 1 (SPTLC1)/SPTLC2, decreased dexamethasone-induced plasma and liver triglyceride levels in WT but not Angptl4-/- mice. We noted similar results in mice infected with adeno-associated virus-expressing small hairpin RNAs targeting Sptlc2. Protein phosphatase 2 phosphatase activator (PP2A) and protein kinase Cζ (PKCζ) are two known downstream effectors of ceramides. We found here that mice treated with an inhibitor of PKCζ, 2-acetyl-1,3-cyclopentanedione (ACPD), had lower levels of dexamethasone-induced triglyceride accumulation in plasma and liver. However, small hairpin RNA-mediated targeting of the catalytic PP2A subunit (Ppp2ca) had no effect on dexamethasone responses on plasma and liver triglyceride levels. Overall, our results indicate that chronic dexamethasone treatment induces an ANGPTL4-ceramide-PKCζ axis that activates hepatic de novo lipogenesis and triglyceride synthesis, resulting in lipid disorders.


Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Ceramidas/metabolismo , Dexametasona/toxicidade , Fígado/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteína 4 Semelhante a Angiopoietina/deficiência , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Hipertrigliceridemia/etiologia , Hipertrigliceridemia/metabolismo , Lipogênese/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase C/antagonistas & inibidores , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismo
15.
Drug Deliv ; 26(1): 237-243, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30883241

RESUMO

Myriocin is an antibiotic derived from Mycelia sterilia, and is a potent inhibitor of serine palmitoyltransferase, the enzyme involved in the first step of sphingosine synthesis. Myriocin, inhibiting ceramide synthesis, has a great potential for treatment of diseases characterized by high ceramide levels in affected tissues, such as retinitis pigmentosa (RP). Drug delivery to the retina is a challenging task, which is generally by-passed through intravitreal injection, that represents a risky invasive procedure. We, therefore, developed and characterized an ophthalmic topical nanotechnological formulation based on a nanostructured lipid carrier (NLC) and containing myriocin. The ocular distribution of myriocin in the back of the eye was assessed both in rabbits and mice using LC-MS/MS. Moreover, rabbit retinal sphingolipid and ceramides levels, after myriocin-NLC (Myr-NLC) eye drops treatment, were assessed. The results demonstrated that Myr-NLC formulation is well tolerated and provided effective levels of myriocin in the back of the eye both in rabbits and mice. We found that Myr-NLC eye drops treatment was able to significantly decrease retinal sphingolipid levels. In conclusion, these data suggest that the Myr-NLC ophthalmic formulation is suitable for pharmaceutical development and warrants further clinical evaluation of this eye drops for the treatment of RP.


Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Soluções Oftálmicas/farmacologia , Retina/efeitos dos fármacos , Retinite Pigmentosa/tratamento farmacológico , Animais , Ceramidas/metabolismo , Química Farmacêutica/métodos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Ácidos Graxos Monoinsaturados/química , Lipídeos/química , Camundongos , Nanoestruturas/química , Soluções Oftálmicas/química , Coelhos , Retina/metabolismo , Retinite Pigmentosa/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo
16.
J Lipid Res ; 60(5): 953-962, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30792183

RESUMO

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.


Assuntos
Serina C-Palmitoiltransferase/metabolismo , Serina/química , Serina/metabolismo , Sphingomonas/enzimologia , Humanos , Marcação por Isótopo , Cinética , Microssomos/enzimologia , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/isolamento & purificação , Especificidade por Substrato
17.
Nat Commun ; 10(1): 813, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30778062

RESUMO

Neurons are highly polarized cells with distinct protein compositions in axonal and dendritic compartments. Cellular mechanisms controlling polarized protein sorting have been described for mature nervous system but little is known about the segregation in newly differentiated neurons. In a forward genetic screen for regulators of Drosophila brain circuit development, we identified mutations in SPT, an evolutionary conserved enzyme in sphingolipid biosynthesis. Here we show that reduced levels of sphingolipids in SPT mutants cause axonal morphology defects similar to loss of cell recognition molecule Dscam. Loss- and gain-of-function studies show that neuronal sphingolipids are critical to prevent aggregation of axonal and dendritic Dscam isoforms, thereby ensuring precise Dscam localization to support axon branch segregation. Furthermore, SPT mutations causing neurodegenerative HSAN-I disorder in humans also result in formation of stable Dscam aggregates and axonal branch phenotypes in Drosophila neurons, indicating a causal link between developmental protein sorting defects and neuronal dysfunction.


Assuntos
Axônios/fisiologia , Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/metabolismo , Esfingolipídeos/metabolismo , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular/genética , Dendritos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Masculino , Corpos Pedunculados/citologia , Corpos Pedunculados/metabolismo , Mutação , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo
18.
J Biol Chem ; 294(13): 5146-5156, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30700557

RESUMO

Sphingolipids compose a lipid family critical for membrane structure as well as intra- and intercellular signaling. De novo sphingolipid biosynthesis is initiated by the enzyme serine palmitoyltransferase (SPT), which resides in the endoplasmic reticulum (ER) membrane. In both yeast and mammalian species, SPT activity is homeostatically regulated through small ER membrane proteins, the Orms in yeast and the ORMDLs in mammalian cells. These proteins form stable complexes with SPT. In yeast, the homeostatic regulation of SPT relies, at least in part, on phosphorylation of the Orms. However, this does not appear to be the case for the mammalian ORMDLs. Here, we accomplished a cell-free reconstitution of the sphingolipid regulation of the ORMDL-SPT complex to probe the underlying regulatory mechanism. Sphingolipid and ORMDL-dependent regulation of SPT was demonstrated in isolated membranes, essentially free of cytosol. This suggests that this regulation does not require soluble cytosolic proteins or small molecules such as ATP. We found that this system is particularly responsive to the pro-apoptotic sphingolipid ceramide and that this response is strictly stereospecific, indicating that ceramide regulates the ORMDL-SPT complex via a specific binding interaction. Yeast membranes harboring the Orm-SPT system also directly responded to sphingolipid, suggesting that yeast cells have, in addition to Orm phosphorylation, an additional Orm-dependent SPT regulatory mechanism. Our results indicate that ORMDL/Orm-mediated regulation of SPT involves a direct interaction of sphingolipid with the membrane-bound components of the SPT-regulatory apparatus.


Assuntos
Ceramidas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Metabolismo dos Lipídeos
19.
Exp Dermatol ; 28 Suppl 1: 43-49, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30698872

RESUMO

BACKGROUND: Pyridoxine (VB6 ), which acts as a coenzyme in the biosynthesis of niacin, is formulated in pharmaceuticals to treat skin roughness. However, the mechanism of action of VB6 is not known precisely. OBJECTIVE: This study was conducted to clarify the influence of highly oxidative conditions on the expression of skin moisture-related mRNAs and to evaluate the preventive effects of VB6 focusing on antioxidant behaviour. METHODS: Intracellular levels of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEKs) were determined using the 2',7'-dichlorofluorescein diacetate assay. Real-time PCR was employed to investigate the influence of higher oxidative conditions on the expression of mRNAs encoding serine palmitoyl transferase (SPT) and filaggrin, and to characterize the mechanism of the antioxidant effect of VB6 . Intracellular glutathione was quantified using an assay based on the glutathione recycling system with 5,5'-dithiobis (2-nitrobenzoic acid) reagent and glutathione reductase. Carbonylated proteins (CPs) were semi-quantified by detecting aldehyde residues. RESULTS: Treatment of NHEKs with BSO increased the level of intracellular CPs by interfering with intracellular glutathione synthesis. Further, treatment with BSO down-regulated the expression level of SPT mRNA, but VB6 restored SPT mRNA expression in BSO-treated NHEKs. VB6 decreased the level of intracellular CPs with or without BSO treatment in a dose-dependent manner. In addition, VB6 increased levels of intracellular NADH/NADPH and glutathione through the activation of nuclear factor E2-related factor 2 (Nrf2) signalling. CONCLUSION: These results suggest that highly oxidative conditions cause an impaired skin barrier function due to the down-regulation of SPT that results in skin roughness. VB6 improved the down-regulation of SPT mRNA expression initiated by highly oxidative conditions by enhancing the intracellular antioxidant system.


Assuntos
Antioxidantes/metabolismo , Oxigênio/metabolismo , Piridoxina/farmacologia , Serina C-Palmitoiltransferase/metabolismo , Pele/efeitos dos fármacos , Regulação para Baixo , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , NAD/metabolismo , NADP/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Niacina/farmacologia , Oxazinas/metabolismo , Estresse Oxidativo , Carbonilação Proteica , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Xantenos/metabolismo
20.
Biochim Biophys Acta Proteins Proteom ; 1867(4): 382-395, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30639427

RESUMO

Sphingolipids are diverse lipids with essential, and occasionally opposing, functions in the cell and therefore tight control over biosynthesis is vital. Mechanisms governing this regulation are not understood. Initial steps in sphingolipid biosynthesis take place on the cytosolic face of the endoplasmic reticulum (ER). Serine palmitoyltransferase (SPT) is an ER-resident enzyme catalyzing the first-committed step in sphingolipid biosynthesis. Not surprisingly, SPT activity is tightly regulated. ORMDLs are ER-resident proteins recently identified as regulators of SPT activity. ORMDL proteins interact directly with SPT but the nature of this interaction is unknown. ORMDL protein sequences contain hydrophobic regions, yet algorithm-based predictions of transmembrane segments are highly ambiguous, making topology of this key regulator unclear. Here we report use of substituted cysteine accessibility to analyze topology of mammalian ORMDLs. We constructed multiple mutant ORMDLs, each containing a single cysteine strategically placed along the protein length. Combined use of selective membrane permeabilization with an impermeant cysteine modification reagent allowed us to assign transmembrane and cytosolic segments of ORMDL. We confirmed that mammalian ORMDL proteins transit the membrane four times, with amino- and carboxy termini facing the cytosol along with a large cytosolic loop. This model will allow us to determine details of the ORMDL-SPT interaction and identify regions acting as the "lipid sensor" to detect changes in cellular sphingolipid levels. We also observe that SPT and ORMDL are substantially resistant to extraction from membranes with non-ionic detergent, indirectly suggesting that both proteins reside in a specialized subdomain of the ER.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Cisteína/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo
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