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1.
J Immunol ; 187(9): 4705-13, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21964029

RESUMO

NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vß8.2 and Vß7 NKT TCR affinity for CD1d-α-C-GalCer, and some related analogs, is ∼10-fold lower than that for the NKT TCR-CD1d-α-GalCer interaction. Nevertheless, the crystal structure of the Vß8.2 NKT TCR-CD1d-α-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.


Assuntos
Antígenos CD1d/metabolismo , Galactosilceramidas/metabolismo , Células T Matadoras Naturais/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Antígenos CD1d/imunologia , Configuração de Carboidratos , Células Cultivadas , Cristalografia por Raios X , Galactosilceramidas/imunologia , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
2.
Carcinogenesis ; 33(9): 1726-35, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22581840

RESUMO

Sphingadienes (SDs) derived from soy and other natural sphingolipids are cytotoxic to colon cancer cells via an Akt-dependent mechanism and reduce adenoma formation in Apc(Min/+) mice. Wnt signaling is fundamental to colon carcinogenesis and is the basis for spontaneous tumorigenesis in Apc(Min/+) mice and patients with familial adenomatous polyposis. In the present study, we investigated the impact of SDs on Wnt signaling. Oral SD administration reduced levels of active ß-catenin and Wnt targets c-Myc and cyclin D1 in Apc(Min/+) mouse intestinal tissues. Colon cancer cells treated with SDs exhibited reduced Wnt transcriptional activity, as well as reduced nuclear ß-catenin localization and subsequent reduction in active-ß-catenin levels. Further, we observed a decrease in phosphorylated (inactive) GSK3ß in SD-treated mice and colon cancer cells. Expression of constitutively active myristoylated-Akt or inactivation of GSK3ß using LiCl attenuated SD-mediated inhibition of Wnt transcriptional activity and active-ß-catenin levels. SDs exhibited additive effects with inhibitors of the phosphatidylinositol-3-kinase/Akt/mTOR pathway to induce cytotoxicity. Further, a combination regime of SDs and low-dose rapamycin decreased visible polyps in Apc(Min/+) mice and reduced the levels of Wnt target gene expression and mTOR target activation. SD-mediated inhibition of Akt and Wnt pathways and cytotoxicity in colon cancer cells was dependent upon the activity of protein phosphatase 2A, as shown by reversal of these effects by pretreatment with okadaic acid or calyculin A. Our cumulative findings indicate that SDs inhibit Wnt signaling through a protein phosphatase 2A/Akt/GSK3ß-dependent mechanism that may contribute to their chemopreventive effects in intestinal tumorigenesis.


Assuntos
Anticarcinógenos/farmacologia , Neoplasias do Colo/prevenção & controle , Quinase 3 da Glicogênio Sintase/fisiologia , Proteína Fosfatase 2/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Esfingolipídeos/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/fisiologia , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Regulação para Baixo , Glicogênio Sintase Quinase 3 beta , Humanos , Elementos de Resposta , Transdução de Sinais , Via de Sinalização Wnt/fisiologia
3.
Biochem Biophys Res Commun ; 424(1): 18-21, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22727907

RESUMO

Sphingosine 1-phosphate, a bioactive signaling molecule with diverse cellular functions, is irreversibly degraded by the endoplasmic reticulum enzyme sphingosine 1-phosphate lyase, generating trans-2-hexadecenal and phosphoethanolamine. We recently demonstrated that trans-2-hexadecenal causes cytoskeletal reorganization, detachment, and apoptosis in multiple cell types via a JNK-dependent pathway. These findings and the known chemistry of related α,ß-unsaturated aldehydes raise the possibility that trans-2-hexadecenal may interact with additional cellular components. In this study, we show that it reacts readily with deoxyguanosine and DNA to produce the diastereomeric cyclic 1,N(2)-deoxyguanosine adducts 3-(2-deoxy-ß-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy-6R-tridecylpyrimido[1,2-a]purine-10(3H)one and 3-(2-deoxy-ß-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy-6S-tridecylpyrimido[1,2-a]purine-10(3H)one. Thus, our findings suggest that trans-2-hexadecenal produced endogenously by sphingosine 1-phosphate lyase can react directly with DNA forming aldehyde-derived DNA adducts with potentially mutagenic consequences.


Assuntos
Aldeídos/química , Adutos de DNA/química , Desoxiguanosina/química , Lisofosfolipídeos/química , Esfingolipídeos/química , Esfingosina/análogos & derivados , Aldeído Liases/química , Lisofosfolipídeos/biossíntese , Mutagênese , Esfingosina/biossíntese , Esfingosina/química
4.
Anal Biochem ; 408(1): 12-8, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20804717

RESUMO

Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography-electrospray ionization-tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25µg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC50 value of 52.4µM.


Assuntos
Aldeído Liases/metabolismo , Aldeídos/análise , Cromatografia Líquida de Alta Pressão/métodos , Lisofosfolipídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Esfingosina/análogos & derivados , Aldeído Liases/antagonistas & inibidores , Animais , Cloridrato de Fingolimode , Hidrogenação , Cinética , Camundongos , Microssomos Hepáticos/enzimologia , Propilenoglicóis/química , Ratos , Semicarbazonas/análise , Esfingosina/química , Esfingosina/metabolismo , Estereoisomerismo , Espectrometria de Massas em Tandem
5.
J Org Chem ; 76(21): 8588-98, 2011 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-21958232

RESUMO

A nonisosteric α-C-glycoside analogue of KRN7000 (α-1C-GalCer, 1) was reported to induce a selective type of cytokine release in human invariant natural killer cells in vitro. We report here a very concise synthetic route to 1 and its analogue 1'. The key steps include olefin cross-metathesis, Sharpless asymmetric epoxidation, and epoxide opening by NaN(3)/NH(4)Cl. Inversion of configuration at the amide-bearing carbon in the phytosphingosine backbone constructed by epoxide opening in our previous synthesis of 1 was verified, indicating that remote group participation is not involved during the epoxide-opening reaction.


Assuntos
Adjuvantes Imunológicos/síntese química , Adjuvantes Imunológicos/farmacologia , Alcenos/química , Compostos de Epóxi/química , Galactosilceramidas/farmacologia , Glicoesfingolipídeos/química , Células T Matadoras Naturais/efeitos dos fármacos , Galactosilceramidas/síntese química , Galactosilceramidas/química , Humanos , Imunização , Conformação Molecular , Estrutura Molecular , Células T Matadoras Naturais/química , Estereoisomerismo
6.
J Exp Med ; 199(6): 763-74, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15007093

RESUMO

Neointimal lesions are characterized by accumulation of cells within the arterial wall and are a prelude to atherosclerotic disease. Here we report that a brief exposure to either alkyl ether analogs of the growth factor-like phospholipid lysophosphatidic acid (LPA), products generated during the oxidative modification of low density lipoprotein, or to unsaturated acyl forms of LPA induce progressive formation of neointima in vivo in a rat carotid artery model. This effect is completely inhibited by the peroxisome proliferator-activated receptor (PPAR)gamma antagonist GW9662 and mimicked by PPARgamma agonists Rosiglitazone and 1-O-hexadecyl-2-azeleoyl-phosphatidylcholine. In contrast, stearoyl-oxovaleryl phosphatidylcholine, a PPARalpha agonist and polypeptide epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor failed to elicit neointima. The structure-activity relationship for neointima induction by LPA analogs in vivo is identical to that of PPARgamma activation in vitro and disparate from that of LPA G protein-coupled receptor activation. Neointima-inducing LPA analogs up-regulated the CD36 scavenger receptor in vitro and in vivo and elicited dedifferentiation of cultured vascular smooth muscle cells that was prevented by GW9662. These results suggest that selected LPA analogs are important novel endogenous PPARgamma ligands capable of mediating vascular remodeling and that activation of the nuclear transcription factor PPARgamma is both necessary and sufficient for neointima formation by components of oxidized low density lipoprotein.


Assuntos
Anilidas/farmacologia , Arteriosclerose/induzido quimicamente , Doenças das Artérias Carótidas/induzido quimicamente , Lisofosfolipídeos/toxicidade , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Análise de Variância , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Células Cultivadas , Primers do DNA , Modelos Animais de Doenças , Substâncias de Crescimento/metabolismo , Ligantes , Lipoproteínas LDL/metabolismo , Masculino , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosiglitazona , Relação Estrutura-Atividade , Tiazolidinedionas/toxicidade , Fatores de Tempo , Fatores de Transcrição/agonistas
7.
J Org Chem ; 75(13): 4356-64, 2010 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-20527744

RESUMO

An asymmetric synthesis of d-ribo-phytosphingosine (1) was achieved by utilizing the ProPhenol (12)-catalyzed alkynylation of unsaturated aldehyde 8 to afford allylic propargylic alcohol (S)-6 followed by asymmetric epoxidation and opening of propargylic epoxy alcohol anti-5 with NaN(3)/NH(4)Cl. Deprotection and reduction of the resulting acyclic azide 3 then gave 1. Alkyne-azide 3 was subjected to an intramolecular click reaction, generating a bicyclic triazole, which was found to have unexpected vicinal coupling constants. Application of the advanced Mosher method verified the configurations of the three contiguous stereogenic centers of 1. An alkynyl azide analogue of 1, which may be useful as a glycosyl acceptor in the synthesis of alpha-galactosylceramide derivatives, was also readily prepared by this route.


Assuntos
Acetaldeído/química , Alcinos/química , Azidas/química , Galactosilceramidas/química , Esfingosina/análogos & derivados , Alquilação , Catálise , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Esfingosina/síntese química , Esfingosina/química , Estereoisomerismo
8.
Biochim Biophys Acta ; 1758(6): 807-12, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16777057

RESUMO

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC

Assuntos
Proteínas de Transporte/metabolismo , Galactosilceramidas/metabolismo , Colesterol/metabolismo , Fosfolipídeos/metabolismo , Esfingomielinas/metabolismo
9.
Chem Phys Lipids ; 194: 2-11, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26187854

RESUMO

We have assessed the effect of two ether glycerol lipids, 77-6 ((2S, 3R)-4-(Tetradecyloxy)-2-amino-1,3-butanediol) and 56-5 ((S)-2-Amino-3-O-hexadecyl-1-propanol), which are substrates for sphingosine kinases, on inflammatory responses. Treatment of differentiated U937 macrophage-like cells with 77-6 but not 56-5 enhanced IL-1ß release; either alone or in the presence of LPS. The stimulatory effect of sphingosine or 77-6 on LPS-stimulated IL-1ß release was reduced by pretreatment of cells with the caspase-1 inhibitor, Ac-YVAD-CHO, thereby indicating a role for the inflammasome. The enhancement of LPS-stimulated IL-1ß release in response to sphingosine, but not 77-6, was reduced by pretreatment of cells with the cathepsin B inhibitor, CA074Me, indicating a role for lysosomal destabilization in the effect of sphingosine. Administration of 56-5 to mice increased disease progression in an experimental autoimmune encephalomyelitis model and this was associated with a considerable increase in the infiltration of CD4(+) T-cells, CD11b(+) monocytes and F4/80(+) macrophages in the spinal cord. 56-5 and 77-6 were without effect on the degradation of myc-tagged sphingosine 1-phosphate 1 receptor in CCL39 cells. Therefore, the effect of 56-5 on EAE disease progression is likely to be independent of the inflammasome or the sphingosine 1-phosphate 1 receptor. However, 56-5 is chemically similar to platelet activating factor and the exacerbation of EAE disease progression might be linked to platelet activating factor receptor signaling.


Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Éteres de Glicerila/farmacologia , Interleucina-1beta/metabolismo , Lipídeos/farmacologia , Animais , Progressão da Doença , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/metabolismo , Éteres de Glicerila/química , Células HEK293 , Humanos , Lipídeos/química , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Esfingosina/farmacologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Células U937
10.
Biochim Biophys Acta ; 1582(1-3): 295-308, 2002 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-12069841

RESUMO

Ligand recognition by G protein-coupled receptors (GPCR), as well as substrate recognition by enzymes, almost always shows a preference for a naturally occurring enantiomer over the unnatural one. Recognition of lysophosphatidic acid (LPA) by its receptors is an exception, as both the natural L (R) and unnatural D (S) stereoisomers of LPA are equally active in bioassays. In contrast to the enantiomers of LPA, analogs of N-acyl-serine phosphoric acid (NASPA) and N-acyl-ethanolamine phosphoric acid (NAEPA), which contain a serine and an ethanolamine backbone, respectively, in place of glycerol, are recognized in a stereoselective manner. This stereoselective interaction may lead to the development of receptor subtype-selective antagonists. In the present study, we review the stereochemical aspects of LPA pharmacology and describe the chemical synthesis of pure LPA enantiomers together with their ligand-binding properties toward the LPA1, LPA2, and LPA3 receptors and their metabolism by lipid phosphate phosphatase 1 (LPP1). Finally, we evaluate the concept of stereopharmacology in developing novel ligands for LPA receptors.


Assuntos
Lisofosfolipídeos/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Animais , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Humanos , Lisofosfolipídeos/química , Lisofosfolipídeos/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores de Ácidos Lisofosfatídicos , Estereoisomerismo
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