RESUMO
Specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins are formed by the consecutive action of 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- or 15-lipoxygenases using arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid as substrate. Lipoxins are trihydroxylated oxylipins which are formed from arachidonic and eicosapentaenoic acid. The latter can also be converted to di- and trihydroxylated resolvins of the E series, whereas docosahexaenoic acid is the substrate for the formation of di- and trihydroxylated resolvins of the D series. Here, we summarize the formation of lipoxins and resolvins in leukocytes. From the data published so far, it becomes evident that FLAP is required for the biosynthesis of most of the lipoxins and resolvins. Even in the presence of FLAP, formation of the trihydroxylated SPMs (lipoxins, RvD1-RvD4, RvE1) in leukocytes is very low or undetectable which is obviously due to the extremely low epoxide formation by 5-LO from oxylipins such as 15-H(p)ETE, 18-H(p)EPE or 17-H(p)DHA. As a result, only the dihydroxylated oxylipins (5 S,15S-diHETE, 5 S,15S-diHEPE) and resolvins (RvD5, RvE2, RvE4) can be consistently detected using leukocytes as SPM source. However, the reported levels of these dihydroxylated lipid mediators are still much lower than those of the typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g. 5-HETE), leukotrienes or cyclooxygenase-derived prostaglandins. Since 5-LO expression is mainly restricted to leukocytes these cells are considered as the main source of SPMs. The low formation of trihydroxylated SPMs in leukocytes, the fact that they are hardly detected in biological samples as well as the lack of functional signaling by their receptors make it highly questionable that trihydroxylated SPMs play a role as endogenous mediators in the resolution of inflammation.
Assuntos
Lipoxinas , Humanos , Lipoxinas/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico , Ácido Araquidônico , Oxilipinas , Eicosanoides/metabolismo , Inflamação/metabolismo , LeucócitosRESUMO
Dicer is a ribonuclease III enzyme in biosynthesis of micro-RNAs (miRNAs). Here we describe a regulation of Dicer expression in monocytic cells, based on proteolysis. In undifferentiated Mono Mac 6 (MM6) cells, full-length Dicer was undetectable; only an â¼50-kDa fragment appeared in Western blots. However, when MM6 cells were treated with zymosan or LPS during differentiation with TGF-ß and 1,25diOHvitD3, full-length Dicer became abundant together with varying amounts of â¼170- and â¼50-kDa Dicer fragments. Mass spectrometry identified the Dicer fragments and showed cleavage about 450 residues upstream from the C terminus. Also, PGE2 (prostaglandin E2) added to differentiating MM6 cells up-regulated full-length Dicer, through EP2/EP4 and cAMP. The TLR stimuli strongly induced miR-146a-5p, while PGE2 increased miR-99a-5p and miR-125a-5p, both implicated in down-regulation of TNFα. The Ser protease inhibitor AEBSF (4-[2-aminoethyl] benzene sulfonyl fluoride) up-regulated full-length Dicer, both in MM6 cells and in primary human blood monocytes, indicating a specific proteolytic degradation. However, AEBSF alone did not lead to a general increase in miR expression, indicating that additional mechanisms are required to increase miRNA biosynthesis. Finally, differentiation of monocytes to macrophages with M-CSF or GM-CSF strongly up-regulated full-length Dicer. Our results suggest that differentiation regimens, both in the MM6 cell line and of peripheral blood monocytes, inhibit an apparently constitutive Dicer proteolysis, allowing for increased formation of miRNAs.
Assuntos
Diferenciação Celular , RNA Helicases DEAD-box/metabolismo , MicroRNAs/metabolismo , Monócitos/metabolismo , Prostaglandina-E Sintases/metabolismo , Proteólise , Ribonuclease III/metabolismo , Células Cultivadas , RNA Helicases DEAD-box/genética , Dinoprostona/farmacologia , Hematopoese , Humanos , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Monócitos/citologia , Monócitos/efeitos dos fármacos , Prostaglandina-E Sintases/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Ribonuclease III/genética , Zimosan/farmacologiaRESUMO
Targeting inflammatory mediators and related signaling pathways may offer a rational strategy for the treatment of cancer. The incorporation of metabolically stable, sterically demanding, and hydrophobic carboranes in dual cycloxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids is a promising approach. The di-tert-butylphenol derivatives R-830, S-2474, KME-4, and E-5110 represent potent dual COX-2/5-LO inhibitors. The incorporation of p-carborane and further substitution of the p-position resulted in four carborane-based di-tert-butylphenol analogs that showed no or weak COX inhibition but high 5-LO inhibitory activities in vitro. Cell viability studies on five human cancer cell lines revealed that the p-carborane analogs R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb exhibited lower anticancer activity compared to the related di-tert-butylphenols. Interestingly, R-830-Cb did not affect the viability of primary cells and suppressed HCT116 cell proliferation more potently than its carbon-based R-830 counterpart. Considering all the advantages of boron cluster incorporation for enhancement of drug biostability, selectivity, and availability of drugs, R-830-Cb can be tested in further mechanistic and in vivo studies.
Assuntos
Boranos , Inibidores de Lipoxigenase , Humanos , Ciclo-Oxigenase 2 , Inibidores de Lipoxigenase/farmacologiaRESUMO
Inhibition of microsomal prostaglandin E synthase-1 (mPGES-1) results in decreased production of proinflammatory PGE2 and can lead to shunting of PGH2 into the prostaglandin D2 (PGD2)/15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) pathway. 15dPGJ2 forms Michael adducts with thiol-containing biomolecules such as GSH or cysteine residues on target proteins and is thought to promote resolution of inflammation. We aimed to elucidate the biosynthesis and metabolism of 15dPGJ2 via conjugation with GSH, to form 15dPGJ2-glutathione (15dPGJ2-GS) and 15dPGJ2-cysteine (15dPGJ2-Cys) conjugates and to characterize the effects of mPGES-1 inhibition on the PGD2/15dPGJ2 pathway in mouse and human immune cells. Our results demonstrate the formation of PGD2, 15dPGJ2, 15dPGJ2-GS, and 15dPGJ2-Cys in RAW264.7 cells after lipopolysaccharide stimulation. Moreover, 15dPGJ2-Cys was found in lipopolysaccharide-activated primary murine macrophages as well as in human mast cells following stimulation of the IgE-receptor. Our results also suggest that the microsomal glutathione S-transferase 3 is essential for the formation of 15dPGJ2 conjugates. In contrast to inhibition of cyclooxygenase, which leads to blockage of the PGD2/15dPGJ2 pathway, we found that inhibition of mPGES-1 preserves PGD2 and its metabolites. Collectively, this study highlights the formation of 15dPGJ2-GS and 15dPGJ2-Cys in mouse and human immune cells, the involvement of microsomal glutathione S-transferase 3 in their biosynthesis, and their unchanged formation following inhibition of mPGES-1. The results encourage further research on their roles as bioactive lipid mediators.
Assuntos
Cisteína , Prostaglandinas , Camundongos , Humanos , Animais , Lipopolissacarídeos/metabolismo , Mastócitos , Prostaglandina-E Sintases/metabolismo , Macrófagos/metabolismo , Ciclo-Oxigenase 2/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Prostaglandina D2/farmacologiaRESUMO
Nuclear receptors (NRs) activate transcription of target genes in response to binding of ligands to their ligand-binding domains (LBDs). Typically, in vitro assays use either gene expression or the recruitment of coactivators to the isolated LBD of the NR of interest to measure NR activation. However, this approach ignores that NRs function as homo- as well as heterodimers and that the LBD harbors the main dimerization interface. Cofactor recruitment is thereby interconnected with oligomerization status as well as ligand occupation of the partnering LBD through allosteric cross talk. Here we present a modular set of homogeneous time-resolved FRET-based assays through which we investigated the activation of PPARγ in response to ligands and the formation of heterodimers with its obligatory partner RXRα. We introduced mutations into the RXRα LBD that prevent coactivator binding but do not interfere with LBD dimerization or ligand binding. This enabled us to specifically detect PPARγ coactivator recruitment to PPARγ:RXRα heterodimers. We found that the RXRα agonist SR11237 destabilized the RXRα homodimer but promoted formation of the PPARγ:RXRα heterodimer, while being inactive on PPARγ itself. Of interest, incorporation of PPARγ into the heterodimer resulted in a substantial gain in affinity for coactivator CBP-1, even in the absence of ligands. Consequently, SR11237 indirectly promoted coactivator binding to PPARγ by shifting the oligomerization preference of RXRα toward PPARγ:RXRα heterodimer formation. These results emphasize that investigation of ligand-dependent NR activation should take NR dimerization into account. We envision these assays as the necessary assay tool kit for investigating NRs that partner with RXRα.
Assuntos
Proteína de Ligação a CREB/metabolismo , PPAR gama/metabolismo , Multimerização Proteica , Receptor X Retinoide alfa/metabolismo , Benzoatos/farmacologia , Células HEK293 , Humanos , Ligantes , Mutação/genética , Coativador 1 de Receptor Nuclear/metabolismo , PPAR gama/agonistas , PPAR gama/química , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Reprodutibilidade dos Testes , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/genética , Retinoides/farmacologia , Rosiglitazona/farmacologia , Ativação Transcricional/genéticaRESUMO
The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Araquidonato 5-Lipoxigenase/genética , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , MicroRNAs/genética , Ribonuclease III/metabolismo , TranscriptomaRESUMO
Endocannabinoids are important lipid-signaling mediators. Both protective and deleterious effects of endocannabinoids in the cardiovascular system have been reported but the mechanistic basis for these contradicting observations is unclear. We set out to identify anti-inflammatory mechanisms of endocannabinoids in the murine aorta and in human vascular smooth muscle cells (hVSMC). In response to combined stimulation with cytokines, IL-1ß and TNFα, the murine aorta released several endocannabinoids, with anandamide (AEA) levels being the most significantly increased. AEA pretreatment had profound effects on cytokine-induced gene expression in hVSMC and murine aorta. As revealed by RNA-Seq analysis, the induction of a subset of 21 inflammatory target genes, including the important cytokine CCL2 was blocked by AEA. This effect was not mediated through AEA-dependent interference of the AP-1 or NF-κB pathways but rather through an epigenetic mechanism. In the presence of AEA, ATAC-Seq analysis and chromatin-immunoprecipitations revealed that CCL2 induction was blocked due to increased levels of H3K27me3 and a decrease of H3K27ac leading to compacted chromatin structure in the CCL2 promoter. These effects were mediated by recruitment of HDAC4 and the nuclear corepressor NCoR1 to the CCL2 promoter. This study therefore establishes a novel anti-inflammatory mechanism for the endogenous endocannabinoid AEA in vascular smooth muscle cells. Furthermore, this work provides a link between endogenous endocannabinoid signaling and epigenetic regulation.
Assuntos
Ácidos Araquidônicos/metabolismo , Quimiocina CCL2/biossíntese , Endocanabinoides/metabolismo , Músculo Liso Vascular/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Ácidos Araquidônicos/farmacologia , Quimiocina CCL2/efeitos dos fármacos , Endocanabinoides/farmacologia , Epigênese Genética/efeitos dos fármacos , Humanos , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Alcamidas Poli-Insaturadas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Sphingosine-1-phosphate (S1P) is involved in the regulation of important cellular processes, including immune-cell trafficking and proliferation. Altered S1P signaling is strongly associated with inflammation, cancer progression, and atherosclerosis; however, the mechanisms underlying its pathophysiologic effects are only partially understood. This study evaluated the effects of S1P in vitro and in vivo on the biosynthesis of leukotrienes (LTs), which form a class of lipid mediators involved in the pathogenesis of inflammatory diseases. Here, we report for the first time that S1P potently suppresses LT biosynthesis in Ca2+-ionophore-stimulated intact human neutrophils. S1P treatment resulted in intracellular Ca2+ mobilization, perinuclear translocation, and finally irreversible suicide inactivation of the LT biosynthesis key enzyme 5-lipoxygenase (5-LO). Agonist studies and S1P receptor mRNA expression analysis provided evidence for a S1P receptor 4-mediated effect, which was confirmed by a functional knockout of S1P4 in HL60 cells. Systemic administration of S1P in wild-type mice decreased both macrophage and neutrophil migration in the lungs in response to LPS and significantly attenuated 5-LO product formation, whereas these effects were abrogated in 5-LO or S1P4 knockout mice. In summary, targeting the 5-LO pathway is an important mechanism to explain S1P-mediated pathophysiologic effects. Furthermore, agonism at S1P4 represents a novel effective strategy in pharmacotherapy of inflammation.-Fettel, J., Kühn, B., Guillen, N. A., Sürün, D., Peters, M., Bauer, R., Angioni, C., Geisslinger, G., Schnütgen, F., Meyer zu Heringdorf, D., Werz, O., Meybohm, P., Zacharowski, K., Steinhilber, D., Roos, J., Maier, T. J. Sphingosine-1-phosphate (S1P) induces potent anti-inflammatory effects in vitro and in vivo by S1P receptor 4-mediated suppression of 5-lipoxygenase activity.
Assuntos
Anti-Inflamatórios/farmacologia , Araquidonato 5-Lipoxigenase/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Receptores de Lisoesfingolipídeo/fisiologia , Esfingosina/análogos & derivados , Animais , Araquidonato 5-Lipoxigenase/biossíntese , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Cálcio/metabolismo , Linhagem Celular , Feminino , Humanos , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Pneumonia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Esfingosina/metabolismo , Esfingosina/farmacologia , Especificidade por SubstratoRESUMO
MicroRNAs (miRs) are important posttranscriptional regulators of gene expression. Besides their well-characterized inhibitory effects on mRNA stability and translation, miRs can also activate gene expression. In this study, we identified a novel noncanonical function of miR-574-5p. We found that miR-574-5p acts as an RNA decoy to CUG RNA-binding protein 1 (CUGBP1) and antagonizes its function. MiR-574-5p induces microsomal prostaglandin E synthase-1 (mPGES-1) expression by preventing CUGBP1 binding to its 3'UTR, leading to an enhanced alternative splicing and generation of an mPGES-1 3'UTR isoform, increased mPGES-1 protein expression, PGE2 formation, and tumor growth in vivo. miR-574-5p-induced tumor growth in mice could be completely inhibited with the mPGES-1 inhibitor CIII. Moreover, miR-574-5p is induced by IL-1ß and is strongly overexpressed in human nonsmall cell lung cancer where high mPGES-1 expression correlates with a low survival rate. The discovered function of miR-574-5p as a CUGBP1 decoy opens up new therapeutic opportunities. It might serve as a stratification marker to select lung tumor patients who respond to the pharmacological inhibition of PGE2 formation.-Saul, M. J., Baumann, I., Bruno, A., Emmerich, A. C., Wellstein, J., Ottinger, S. M., Contursi, A., Dovizio, M., Donnini, S., Tacconelli, S., Raouf, J., Idborg, H., Stein, S., Korotkova, M., Savai, R., Terzuoli, E., Sala, G., Seeger, W., Jakobsson, P.-J., Patrignani, P., Suess, B., Steinhilber, D. miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.
Assuntos
Proteínas CELF1/metabolismo , MicroRNAs/metabolismo , Prostaglandina-E Sintases/metabolismo , RNA/metabolismo , Células A549 , Animais , Proteínas CELF1/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , Mimetismo Molecular , Neoplasias Experimentais , Prostaglandina-E Sintases/genética , Ligação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , RNA/genética , Interferência de RNA , Isoformas de RNA , RNA MensageiroRESUMO
The enzyme 5-lipoxygenase (5-LO) is key in the synthesis of leukotrienes, which are potent proinflammatory lipid mediators involved in chronic inflammatory diseases including cancer. 5-LO is expressed in immune cells but also found in cancer cells. Although the role of 5-LO in tumor cells is beginning to emerge, with the notion that tumor-promoting functions are attributed to its products, the function of 5-LO in the tumor microenvironment remains unclear. To understand the role of 5-LO and its products in the tumor microenvironment, we analyzed its expression and function in tumor-associated macrophages (TAMs). TAMs were generated by coculturing primary human macrophages (MΦ) with human MCF-7 breast carcinoma cells, which caused cell death of cancer cells followed by phagocytosis of cell debris by MΦ. Expression and activity of 5-LO in TAMs were reduced upon coculture with cancer cells. Downregulation of 5-LO in TAMs required tumor cell death and the direct contact between MΦ and dying cancer cells via Mer tyrosine kinase. Subsequently, upregulation of proto-oncogene c-Myb in TAMs induced a stable transcriptional repression of 5-LO. Reduced 5-LO expression in TAMs was mechanistically coupled to an attenuated T cell recruitment. In primary TAMs from human and murine breast tumors, 5-LO expression was absent or low when compared with monocyte-derived MΦ. Our data reveal that 5-LO, which is required for leukotriene production and subsequent T cell recruitment, is downregulated in TAMs through Mer tyrosine kinase-dependent recognition of apoptotic cancer cells. Mechanistically, we noticed transcriptional repression of 5-LO by proto-oncogene c-Myb and conclude that loss of stromal 5-LO expression favors tumor progression.
Assuntos
Apoptose , Araquidonato 5-Lipoxigenase/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Araquidonato 5-Lipoxigenase/genética , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Cultivadas , Quimiotaxia de Leucócito/imunologia , Ativação Enzimática , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Macrófagos/patologia , Camundongos , Neoplasias/genética , Neoplasias/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myb/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transcrição GênicaRESUMO
Eleven new 4-(4-chlorophenyl)thiazol-2-amines were synthesized and, together with nine known derivatives, evaluated in vitro for inhibitory properties towards bovine pancreatic DNase I. Three compounds (18-20) inhibited DNase I with IC50 values below 100 µM, with compound 19 being the most potent (IC50 = 79.79 µM). Crystal violet, used as a positive control in the absence of a "golden standard", exhibited almost 5-fold weaker DNase I inhibition. Pharma/E-State RQSAR models clarified critical structural fragments relevant for DNase I inhibition. Molecular docking and molecular dynamics simulation defined the 4-(4-chlorophenyl)thiazol-2-amines interactions with the most important catalytic residues of DNase I. Ligand-based pharmacophore modeling and virtual screening confirmed the chemical features of 4-(4-chlorophenyl)thiazol-2-amines required for DNase I inhibition and proved the absence of structurally similar molecules in available databases. Compounds 18-20 have been shown as very potent 5-LO inhibitors with nanomolar IC50 values obtained in cell-free assay, with compound 20 being the most potent (IC50 = 50 nM). Molecular docking and molecular dynamics simulations into the binding site of 5-LO enzyme allowed us to clarify the binding mode of these dual DNase I/5-LO inhibitors. It was shown that compounds 18-20 uniquely show interactions with histidine residues in the catalytic site of DNase I and 5-LO enzyme. In the absence of potent organic DNase I inhibitors, compounds 18-20 represent a good starting point for the development of novel Alzheimer's therapeutics based on dual 5-LO and DNase I inhibition, which also have anti-inflammatory properties.
Assuntos
Aminas/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Desoxirribonuclease I/antagonistas & inibidores , Inibidores de Lipoxigenase/uso terapêutico , Doenças Neurodegenerativas/tratamento farmacológico , Tiazóis/química , Aminas/química , Aminas/farmacologia , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Humanos , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacocinética , Inibidores de Lipoxigenase/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Relação Quantitativa Estrutura-AtividadeRESUMO
RATIONALE: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of covalent 5-lipoxygenase inhibitors is challenging due to unknown amino acid specificity and low posttranslational modification (PTM)-identification rates. The analysis of the amino-acid specificity and of the characteristic fragmentation of chemically modified peptides is considered to improve knowledge for the analysis of chemically modified peptides and proteins by MALDI-MS. METHODS: Various compounds were used to investigate the modification of synthetic peptides carrying reactive amino acid residues. Mass spectra were recorded using a MALDI-LTQ Orbitrap XL for high-resolution mass spectrometry and ion trap MALDI-MS2 . UV-Vis-based reduction and radical scavenging analysis was conducted. The on-plate digestion method described by Rühl et al was utilized for modification-site analysis at 5-lipoxygenase. RESULTS: The analysis of amino-acid-specific reactivity revealed the reactivity of quinones towards cysteine residues and the potential occurrence of a subsequent oxidative process was observed by an UV-Vis-based reduction assay. MALDI collision-induced dissociation tandem mass spectrometry (CID-MS2 ) indicated a prominent fragmentation mechanism of modified cysteine and histidine residues. Fragmentation included highly abundant neutral-loss signals which could be used to identify new modifications induced by chemical modifiers at the cysteine-159 residue of 5-lipoxygenase. CONCLUSIONS: Specificity and fragmentation analysis provides crucial information for the analysis of chemically modified cysteines and histidines by MALDI-MS. Elucidation of binding sites by MALDI-MS has been significantly improved using an easy-to-run peptide assay and gives background information for the analysis in the case of chemically modified 5-lipoxygenase.
Assuntos
Peptídeos/química , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Sítios de Ligação , Cisteína/análise , Cisteína/química , Cisteína/metabolismo , Histidina/análise , Histidina/química , Histidina/metabolismo , Lipoxigenase , Inibidores de Lipoxigenase , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Peptídeos/análise , Peptídeos/metabolismo , Ligação Proteica , Proteínas/análise , Proteínas/metabolismo , Quinonas/químicaRESUMO
5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes and specialized proresolving lipid mediators (SPM). It is mainly expressed in leukocytes and is part of the innate immune system. 5-LO can shuttle between the cytosol and the nucleus. Upon cell activation the protein translocates from soluble cellular compartments to the nuclear membrane. Besides FLAP which is required for cellular leukotriene and SPM formation, 5-LO interacts with other proteins like coactosin-like protein (CLP), Dicer, ß-catenin and p53. In this review, the factors involved in the regulation of 5-LO expression, the role of 5-LO in the regulation of stem cell proliferation and differentiation and its biological functions apart from leukotriene and SPM formation are summarized.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Leucotrienos/biossíntese , Animais , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização WntRESUMO
Peroxisome proliferator-activated receptor gamma (PPARγ) modulators have found wide application for the treatment of cancers, metabolic disorders and inflammatory diseases. Contrary to PPARγ agonists, PPARγ antagonists have been much less studied and although they have shown immunomodulatory effects, there is still no therapeutically useful PPARγ antagonist on the market. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), the recently described (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB, T-10017) is a promising prototype for a new class of PPARγ antagonists. It exhibits competitive antagonism against rosiglitazone mediated activation of PPARγ ligand binding domain (PPARγLBD) in a transactivation assay in HEK293T cells with an IC50 of 4.3⯵M against 1⯵M rosiglitazone. The aim of this study was to investigate the structure-activity relationships (SAR) of the MTTB scaffold focusing on improving its physicochemical properties. Through this optimization, 34 new derivatives were prepared and characterized. Two new potent compounds (T-10075 and T-10106) with much improved drug-like properties and promising pharmacokinetic profile were identified.
Assuntos
Cinamatos/farmacologia , PPAR gama/antagonistas & inibidores , Quinolinas/farmacologia , Animais , Cinamatos/síntese química , Cinamatos/farmacocinética , Células HEK293 , Humanos , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/farmacocinética , Ratos , Rosiglitazona/farmacologia , Relação Estrutura-AtividadeRESUMO
Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene.
Assuntos
Proteína p300 Associada a E1A/genética , Histona Desacetilase 1/genética , Prostaglandina-E Sintases/genética , Transcrição Gênica/efeitos dos fármacos , Acetilação , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Dinoprostona/biossíntese , Dinoprostona/genética , Proteína p300 Associada a E1A/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/administração & dosagem , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Camundongos , Prostaglandina-E Sintases/biossíntese , Processamento de Proteína Pós-Traducional/genética , VorinostatRESUMO
Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Núcleo Celular/ultraestrutura , Citosol/ultraestrutura , Isoformas de Proteínas/metabolismo , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/isolamento & purificação , Núcleo Celular/metabolismo , Citosol/metabolismo , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Leucotrienos/biossíntese , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificaçãoRESUMO
5-Lipoxygenase (5-LO) catalyzes the initial two steps of the conversion of arachidonic acid to leukotrienes which represent a group of pro-inflammatory lipid mediators involved in immune defense reactions as well as inflammation, allergy and cancer. Transforming growth factor-ß (TGFß) and calcitriol strongly upregulate 5-LO expression during myeloid cell differentiation and MLL-AF4 has been shown to strongly activate the 5-LO promoter. Here, we investigated the role of TGFß/SMAD signalling in 5-LO promoter activation. We identified two functional SMAD binding elements in the proximal part of the 5-LO promoter which significantly induce 5-LO promoter activity via TGFß and SMAD3/4. Since aberrant 5-LO gene expression has been linked with mixed lineage leukemia (MLL) which is characterized by the presence of MLL fusion proteins (e.g. MLL-AF4), we also investigated the influence of TGFß/SMADs on MLL- and MLL-AF4-mediated 5-LO promoter activation. Our data show that induction of 5-LO promoter activity by SMAD3/4 is MLL-dependent and that knockdown of the MLL complex component MEN1 attenuates the SMAD effect. Our data suggest that induction of 5-LO gene expression by TGFß is at least in part due to stimulation of transcript initiation.
Assuntos
Araquidonato 5-Lipoxigenase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas/genética , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Araquidonato 5-Lipoxigenase/biossíntese , Sequência de Bases , Sítios de Ligação , Indução Enzimática , Técnicas de Silenciamento de Genes , Células HeLa , HumanosRESUMO
5-Lipoxygenase (5-LO) is the key enzyme in leukotriene biosynthesis. Leukotrienes are mediators of the innate immune system and inflammatory processes, and they might also be involved in cancer development. MicroRNAs (miRNAs) are important translational regulators and have been shown to be involved in development, differentiation, and cancer. Unraveling the miRNA network is important for understanding the cellular regulation processes. We identified two new miRNAs, miR-19a-3p and miR-125b-5p, regulating 5-LO and confirmed direct interaction by reporter gene assays. Furthermore, we investigated the regulation of 5-LO by these two miRNAs in several cell types. Inhibition of both miRNAs by antagomirs during differentiation of the myeloid cell line Mono Mac 6 led to a significant increase in 5-LO protein expression. Stimulation of human T lymphocytes with PHA resulted in a strong downregulation of 5-LO mRNA expression and in the induction of miR-19a-3p. The inhibition of miR-19a-3p with an antagomir led to a significant increase in 5-LO mRNA expression in T lymphocytes. Taken together, our data reveal that miR-19a-3p and miR-125b-5p target 5-LO in a cell type- and stimulus-specific manner.
Assuntos
Araquidonato 5-Lipoxigenase/biossíntese , Regulação da Expressão Gênica/imunologia , MicroRNAs/imunologia , Animais , Araquidonato 5-Lipoxigenase/genética , Western Blotting , Células COS , Linhagem Celular , Separação Celular , Chlorocebus aethiops , Citometria de Fluxo , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase em Tempo RealRESUMO
5-Lipoxygenase (5LO) is a key enzyme in leukotriene (LT) biosynthesis. Two accessory proteins, coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP), can support 5LO activity. To study the roles of CLP and FLAP, we knocked down these proteins in the human monocytic cell line Mono Mac 6 (MM6). Expression of CLP increased MM6 cellular 5LO activity for all stimuli tested. CLP is not absolutely crucial, however; some 5LO activity remained in all incubations of CLP knockdown cells. FLAP knockdown had minor effects in the presence of exogenous arachidonic acid, but led to prominent reductions in 5LO product formation from endogenous substrate. Similar effects were observed after CLP and FLAP knockdown in human primary macrophages as well. In addition, FLAP knockdown reduced conversion of leukotriene A4 to leukotriene C4 (LTC4), suggesting a role for the activity of LTC4 synthase. After stimulation of MM6 cells by phorbol myristate acetate and ionophore A23187, a perinuclear ring pattern was observed for 5LO. This redistribution from cytosolic to perinuclear was clearly compromised in both CLP- and FLAP-deficient cells. In addition, association of CLP with the nucleus was almost absent after 5LO knockdown, and was clearly reduced in FLAP knockdown cells. Coimmunoprecipitation experiments indicated that 5LO-CLP complex formation in MM6 cells was increased by stimulation with ionophore, and that this complex was formed to the same extent in FLAP knockdown cells. A possible interpretation of our findings is that on cell stimulation, formation of the 5LO-CLP complex augments the translocation from cytosol to nucleus, whereas FLAP stabilizes association of this complex with the perinuclear membrane.
Assuntos
Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Leucotrienos/biossíntese , Proteínas dos Microfilamentos/metabolismo , Monócitos/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/farmacologia , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Imunoprecipitação , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Ionóforos/farmacologia , Leucotrieno C4/biossíntese , Modelos Biológicos , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Ligação Proteica/efeitos dos fármacosRESUMO
5-Lipoxygenase (5-LOX) catalyzes two steps in the biosynthesis of leukotrienes (LTs), lipid mediators of inflammation derived from arachidonic acid. In this review we focus on 5-LOX biochemistry including 5-LOX interacting proteins and regulation of enzyme activity. LTs function in normal host defense, and have roles in many disease states where acute or chronic inflammation is part of the pathophysiology, as briefly summarized at the end of this chapter. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".