RESUMO
The cold shock protein Y-box (YB) binding-1 is an example of a highly regulated protein with pleiotropic functions. Besides activities as a transcription factor in the nucleus or regulator of translation in the cytoplasm, recent findings indicate extracellular effects and secretion via a non-classical secretion pathway. This review summarizes regulatory pathways in which YB-1 participates, all iterating auto-regulatory loops. Schematics are developed that elucidate the cold shock protein activities in (i) fine-tuning its own expression level following platelet-derived growth factor-B-, thrombin- or interferon-γ-dependent signaling, (ii) as a component of the messenger ribonucleoprotein (mRNP) complex for interleukin-2 synthesis in T-cell commitment/activation, (iii) pro-fibrogenic cell phenotypic changes mediated by transforming growth factor-ß, and (iv) receptor Notch-3 cleavage and signal transduction. Emphasis is put forward on subcellular protein translocation mechanisms and underlying signaling pathways. These have mostly been analysed in cell culture systems and rarely in experimental models. In sum, YB-1 seems to fulfill a pacemaker role in diverse diseases, both inflammatory/pro-fibrogenic as well as tumorigenic. A clue towards potential intervention strategies may reside in the understanding of the outlined auto-regulatory loops and means to interfere with cycling pathways.
Assuntos
Proteína 1 de Ligação a Y-Box/metabolismo , Comunicação Celular/fisiologia , Humanos , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Neutral lipid storage in lipid droplets (LDs) is a conserved process across diverse species. Although significant attention has focused on LDs in the biology of obesity, diabetes, and atherosclerosis, there is limited information on the role of LDs in pathogenic fungi. We have disrupted the Fat storage-Inducing Transmembrane (FIT) protein 2 genes of the emerging pathogenic fungus Candida parapsilosis and demonstrated that LD formation is significantly reduced in the mutant cells. Disruption of FIT2 genes also reduced accumulation of triacylglycerols. The production of other lipids such as phospholipids and steryl esters were also affected in the mutant strain. Inhibition of de novo fatty acid biosynthesis by triclosan in the FIT2 disruptants reduced fungal growth in rich medium YPD, indicating that TAGs or fatty acids from the LDs could be important for cell proliferation. FIT2 disruption was associated with enhanced sensitivity to oxidative stress. Furthermore, we showed that FIT2 deletion yeast cells were significantly attenuated in murine infection models, suggesting an involvement of LDs in the pathobiology of the fungus.
Assuntos
Candida/genética , Candida/patogenicidade , Proteínas Fúngicas/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Antifúngicos/metabolismo , Ácidos Graxos/metabolismo , Feminino , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos A , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triclosan/metabolismo , Triglicerídeos/metabolismo , VirulênciaRESUMO
The ability to store fat in the form of cytoplasmic triglyceride droplets is conserved from Saccharomyces cerevisiae to humans. Although much is known regarding the composition and catabolism of lipid droplets, the molecular components necessary for the biogenesis of lipid droplets have remained obscure. Here we report the characterization of a conserved gene family important for lipid droplet formation named fat-inducing transcript (FIT). FIT1 and FIT2 are endoplasmic reticulum resident membrane proteins that induce lipid droplet accumulation in cell culture and when expressed in mouse liver. shRNA silencing of FIT2 in 3T3-LI adipocytes prevents accumulation of lipid droplets, and depletion of FIT2 in zebrafish blocks diet-induced accumulation of lipid droplets in the intestine and liver, highlighting an important role for FIT2 in lipid droplet formation in vivo. Together these studies identify and characterize a conserved gene family that is important in the fundamental process of storing fat.
Assuntos
Tecido Adiposo/fisiologia , Evolução Molecular , Regulação da Expressão Gênica , Fígado/metabolismo , Proteínas de Membrana/fisiologia , Células 3T3-L1/metabolismo , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Diabetes Mellitus Experimental/metabolismo , Humanos , Lipídeos/química , Proteínas de Membrana/química , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Triglicerídeos/química , Triglicerídeos/metabolismo , Peixe-ZebraAssuntos
Aldosterona/metabolismo , Hipertensão/tratamento farmacológico , Hipoaldosteronismo/tratamento farmacológico , Complicações Cardiovasculares na Gravidez/tratamento farmacológico , Cloreto de Sódio/farmacologia , Adulto , Pressão Sanguínea , Feminino , Humanos , Gravidez , Renina/metabolismoRESUMO
Hypertension and sodium retention are features of a diminished 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). The activity of this enzyme is reduced in various disease states with abnormal renal sodium retention and hypertension, including preeclampsia. ATP release to the extracellular compartment is observed with shear stress, inflammation, and placental ischemia. It was hypothesized that ATP downregulates 11beta-HSD2 activity. For that purpose, cell lines from different tissues that previously were used to study the regulation of 11beta-HSD2 were investigated: JEG-3, a vascular trophoblastic; LLCPK1, a renal tubular; and SW620, a colonic epithelial cell line. The 11beta-HSD2 activity, assessed by the conversion of 3H-cortisol to cortisone, was reversibly reduced during incubation with ATP or its stable analogue ATPgammaS in intact JEG-3 and LLCPK1, but not in SW620 cells. In JEG-3 cells, the purinergic antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid but not suramin reversed the inhibition. Incubation with UTP and ADP and their degradation products including adenosine and alpha,beta-methylene-ATP did not inhibit 11beta-HSD2 activity. In contrast, 11beta-HSD2 activity increased almost 2.5-fold after incubation with 2'-methylthio-ATP. This indicates a bidirectional regulation by nucleotides via purinergic receptors. In JEG-3 cells, ATP/ATPgammaS did not alter 11beta-HSD2 promoter activity but reduced 11beta-HSD2 protein and mRNA concentration and half-life, suggesting a posttranscriptional regulation. In conclusion, ATP inhibits cell type specifically via purinergic receptors the expression and activity of the 11beta-HSD2 by a posttranscriptional mechanism.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/efeitos dos fármacos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Trifosfato de Adenosina/farmacologia , Células Epiteliais/metabolismo , Receptores Purinérgicos , Sódio/metabolismo , Western Blotting , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/citologia , Feminino , Humanos , Hipertensão Induzida pela Gravidez/enzimologia , Hipertensão Induzida pela Gravidez/fisiopatologia , Mucosa Intestinal/citologia , Túbulos Renais/citologia , Gravidez , RNA Mensageiro/análise , Valores de Referência , Sensibilidade e Especificidade , Trofoblastos/citologiaRESUMO
11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1), catalyzing the intracellular activation of cortisone to cortisol, is currently considered a promising target to treat patients with metabolic syndrome; hence, there is considerable interest in the development of selective inhibitors. For preclinical tests of such inhibitors, the characteristics of 11beta-HSD1 from the commonly used species have to be known. Therefore, we determined differences in substrate affinity and inhibitor effects for 11beta-HSD1 from six species. The differences in catalytic activities with cortisone and 11-dehydrocorticosterone were rather modest. Human, hamster and guinea-pig 11beta-HSD1 displayed the highest catalytic efficiency in the oxoreduction of cortisone, while mouse and rat showed intermediate and dog the lowest activity. Murine 11beta-HSD1 most efficiently reduced 11-dehydrocorticosterone, while the enzyme from dog showed lower activity than those from the other species. 7-ketocholesterol (7KC) was stereospecifically converted to 7beta-hydroxycholesterol by recombinant 11beta-HSD1 from all species analyzed except hamster, which showed a slight preference for the formation of 7alpha-hydroxycholesterol. Importantly, guinea-pig and canine 11beta-HSD1 displayed very low 7-oxoreductase activities. Furthermore, we demonstrate significant species-specific variability in the potency of various 11beta-HSD1 inhibitors, including endogenous compounds, natural chemicals and pharmaceutical compounds. The results suggest significant differences in the three-dimensional organization of the hydrophobic substrate-binding pocket of 11beta-HSD1, and they emphasize that species-specific variability must be considered in the interpretation of results obtained from different animal experiments. The assessment of such differences, by cell-based test systems, may help to choose the appropriate animal for safety and efficacy studies of novel potential drug candidates.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico , Linhagem Celular , Clonagem Molecular , Corticosterona/análogos & derivados , Corticosterona/metabolismo , Cortisona/metabolismo , Cricetinae , DNA Complementar/genética , Cães , Inibidores Enzimáticos/farmacologia , Cobaias , Humanos , Técnicas In Vitro , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
BACKGROUND: In normal pregnancy, an increased aldosterone (Aldo) concentration coincides with volume expansion. In preeclampsia, Aldo levels are low despite intravascular volume depletion. The present investigation aimed to characterize the compromised Aldo synthesis in preeclampsia, and to identify the molecular basis hereof. METHODS: We recruited 66 pregnant women (24 uneventful, 42 preeclamptic). Genomic DNA was isolated from peripheral blood leukocytes. Urine samples were obtained for gas chromatography-mass spectroscopic measurements of steroid hormones reflecting apparent Aldo synthase (CYP11B2) and 11-hydroxylase (CYP11B1) activities. Polymerase chain reaction (PCR)-based screening for CYP11B2 mutations was performed by SSCP, restriction analysis, and sequencing. RESULTS: CYP11B1 activity was unaltered, but reduction of mean tetrahydro (TH)-Aldo excretion by a factor of 3.9 indicated a diminished CYP11B2 activity in preeclampsia. Accordingly, the ratios of (TH-11-dehydrocorticosterone [A]+TH-corticosterone [B]+5alpha-THB) to (TH-cortisone +TH-cortisol [F]+5alpha-THF) and of 18-OH-THA to THAldo were increased in preeclampsia 2.6- and 15.2-fold, respectively, indicating reduced Aldo synthesis due to diminished methyl oxidase (MO) activity. A lower percentage of women with normal pregnancies had CYP11B2 mutations when compared to preeclamptic women (P < 0.05). Eight polymorphisms were detected, two of which were non-amino acid conserving. Of those, the mutation V386A, earlier found to jeopardize MO activity, was exclusively observed in preeclampsia (0% vs. 17%; P < 0.05). CONCLUSION: Aldo deficiency due to a compromised MO step of Aldo synthesis favors extracellular volume depletion, and may account for an increased risk of placental hypoperfusion and consecutive development of preeclampsia. The sole presence of mutation V386A in preeclamptic mothers may identify a subgroup with an increased risk to develop preeclampsia during pregnancy.
Assuntos
Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Polimorfismo Conformacional de Fita Simples , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Adulto , Creatinina/urina , Ativação Enzimática , Feminino , Humanos , Leucócitos/fisiologia , Gravidez , Esteroide 11-beta-Hidroxilase/genética , Esteroide 11-beta-Hidroxilase/metabolismo , Esteroides/urinaRESUMO
BACKGROUND: In preeclampsia, cortisol degradation by the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is compromised, which enhances intracellular cortisol availability. This leads to vasoconstriction and renal sodium retention with volume expansion, thus increasing blood pressure. An augmented availability of angiotensin II (Ang II) predisposes to preeclampsia. Some effects of Ang II are mediated by the mitogen-activated protein kinase (MAPK) cascade, which also regulates 11beta-HSD2 activity. Therefore, we hypothesized that Ang II regulates 11beta-HSD2. METHODS: The human choriocarcinoma cell line JEG-3, which expresses the 11beta-HSD2 isoenzyme, was used. 3H-cortisol/cortisone conversion assays and mRNA analyses by reverse transcription-polymerase chain reaction (RT-PCR) were performed. Cells were stimulated with Ang II and the effect was modulated by Ang II type 1 (AT1) and AT2 receptor blockers DUP753 or L-158809 and PD-123319, respectively. In order to elucidate the signaling cascade, the MAPK kinase inhibitors PD-098059 and U-0126 were probed. The impact of a modulated 11beta-HSD2 activity was assessed by determining the effect of cortisol on AT1 receptor mRNA. RESULTS: Ang II reduced mRNA and activity of 11beta-HSD2 mainly by a post-transcriptional mechanism. This Ang II effect was abrogated by AT2, but not by AT1 receptor blockade. Mitogen-activated protein (MAP) kinase kinase (MAPKK) inhibitors reversed the Ang II effect. Dexamethasone augmented the mRNA expression of AT1 receptors. Cortisol enhanced AT1 receptor mRNA expression when the 11beta-HSD2 activity was reduced either by Ang II or by glycyrrhetinic acid, an 11beta-HSD2 inhibitor. CONCLUSION: Ang II decreases the activity of 11beta-HSD2 by an AT2 receptor- and MAPK-dependent mechanism. The decreased activity of 11beta-HSD2 increases the intracellular availability of cortisol, which might be relevant for the pathogenesis of hypertension and preeclampsia.