RESUMO
Depletion of exogenous inositol in yeast results in rising levels of phosphatidic acid (PA) and is correlated with increased expression of genes containing the inositol-dependent upstream activating sequence promoter element (UASINO). INO1, encoding myo-inositol 3-phosphate synthase, is the most highly regulated of the inositol-dependent upstream activating sequence-containing genes, but its mechanism of regulation is not clear. In the current study, we determined the relative timing and kinetics of appearance of individual molecular species of PA following removal of exogenous inositol in actively growing wild type, pah1Δ, and ole1ts strains. We report that the pah1Δ strain, lacking the PA phosphatase, exhibits a delay of about 60 min in comparison to wildtype before initiating derepression of INO1 expression. The ole1ts mutant on the other hand, defective in fatty acid desaturation, when grown at a semirestrictive temperature, exhibited reduced synthesis of PA species 34:1 and elevated synthesis of PA species 32:1. Importantly, we found these changes in the fatty acid composition in the PA pool of the ole1ts strain were associated with reduced expression of INO1, indicating that synthesis of PA 34:1 is involved in optimal expression of INO1 in the absence of inositol. Using deuterium-labeled glycerol in short-duration labeling assays, we found that changes associated with PA species 34:1 were uniquely correlated with increased expression of INO1 in all three strains. These data indicate that the signal for activation of INO1 transcription is not necessarily the overall level of PA but rather levels of a specific species of newly synthesized PA 34:1.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ácidos Graxos/metabolismo , Inositol/metabolismo , Ácidos Fosfatídicos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In the yeast Saccharomyces cerevisiae, the Opi1p repressor controls the expression of INO1 via the Opi1p/Ino2p-Ino4p regulatory circuit. Inositol depletion favors Opi1p interaction with both Scs2p and phosphatidic acid at the endoplasmic reticulum (ER) membrane. Inositol supplementation, however, favors the translocation of Opi1p from the ER into the nucleus, where it interacts with the Ino2p-Ino4p complex, attenuating transcription of INO1 A strain devoid of Scs2p (scs2Δ) and a mutant, OPI1FFAT, lacking the ability to interact with Scs2p were utilized to examine the specific role(s) of the Opi1p-Scs2p interaction in the regulation of INO1 expression and overall lipid metabolism. Loss of the Opi1p-Scs2p interaction reduced INO1 expression and conferred inositol auxotrophy. Moreover, inositol depletion in strains lacking this interaction resulted in Opi1p being localized to sites of lipid droplet formation, coincident with increased synthesis of triacylglycerol. Supplementation of choline to inositol-depleted growth medium led to decreased TAG synthesis in all three strains. However, in strains lacking the Opi1p-Scs2p interaction, Opi1p remained in the nucleus, preventing expression of INO1 These data support the conclusion that a specific pool of phosphatidic acid, associated with lipid droplet formation in the perinuclear ER, is responsible for the initial rapid exit of Opi1p from the nucleus to the ER and is required for INO1 expression in the presence of choline. Moreover, the mitochondria-specific phospholipid, cardiolipin, was significantly reduced in both strains compromised for Opi1p-Scs2p interaction, indicating that this interaction is required for the transfer of phosphatidic acid from the ER to the mitochondria for cardiolipin synthesis.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Ácidos Fosfatídicos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Cardiolipinas/metabolismo , Núcleo Celular/metabolismo , Colina/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Gotículas Lipídicas , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Mutação , Mio-Inositol-1-Fosfato Sintase/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Depriving wild type yeast of inositol, a soluble precursor for phospholipid, phosphoinositide, and complex sphingolipid synthesis, activates the protein kinase C (PKC)-MAPK signaling pathway, which plays a key role in the activation of NAD(+)-dependent telomeric silencing. We now report that triggering PKC-MAPK signaling by inositol deprivation or by blocking inositol-containing sphingolipid synthesis with aureobasidin A results in increased telomeric silencing regulated by the MAPK, Slt2p, and the NAD(+)-dependent deacetylase, Sir2p. Consistent with the dependence on NAD(+) in Sir2p-regulated silencing, we found that inositol depletion induces the expression of BNA2, which is required for the de novo synthesis of NAD(+). Moreover, telomeric silencing is greatly reduced in bna2Δ and npt1Δ mutants, which are defective in de novo and salvage pathways for NAD(+) synthesis, respectively. Surprisingly, however, omitting nicotinic acid from the growth medium, which reduces cellular NAD(+) levels, leads to increased telomeric silencing in the absence of inositol and/or at high temperature. This increase in telomeric silencing in response to inositol starvation is correlated to chronological life span extension but is Sir2p-independent. We conclude that activation of the PKC-MAPK signaling by interruption of inositol sphingolipid synthesis leads to increased Sir2p-dependent silencing and is dependent upon the de novo and salvage pathways for NAD(+) synthesis but is not correlated with cellular NAD(+) levels.
Assuntos
Inositol/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase C/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Telômero/ultraestrutura , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Genes Reporter , Mutação , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , TemperaturaRESUMO
The protein kinase C (PKC)-MAPK signaling cascade is activated and is essential for viability when cells are starved for the phospholipid precursor inositol. In this study, we report that inhibiting inositol-containing sphingolipid metabolism, either by inositol starvation or treatment with agents that block sphingolipid synthesis, triggers PKC signaling independent of sphingoid base accumulation. Under these same growth conditions, a fluorescent biosensor that detects the necessary PKC signaling intermediate, phosphatidylinositol (PI)-4-phosphate (PI4P), is enriched on the plasma membrane. The appearance of the PI4P biosensor on the plasma membrane correlates with PKC activation and requires the PI 4-kinase Stt4p. Like other mutations in the PKC-MAPK pathway, mutants defective in Stt4p and the PI4P 5-kinase Mss4p, which generates phosphatidylinositol 4,5-bisphosphate, exhibit inositol auxotrophy, yet fully derepress INO1, encoding inositol-3-phosphate synthase. These observations suggest that inositol-containing sphingolipid metabolism controls PKC signaling by regulating access of the signaling lipids PI4P and phosphatidylinositol 4,5-bisphosphate to effector proteins on the plasma membrane.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Membrana Celular/metabolismo , Inositol/química , Proteína Quinase C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Esfingolipídeos/química , Técnicas Biossensoriais , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Fenótipo , Transdução de Sinais , Temperatura , Fatores de TempoRESUMO
The emerging field of lipidomics, driven by technological advances in lipid analysis, provides greatly enhanced opportunities to characterize, on a quantitative or semi-quantitative level, the entire spectrum of lipids, or lipidome, in specific cell types. When combined with advances in other high throughput technologies in genomics and proteomics, lipidomics offers the opportunity to analyze the unique roles of specific lipids in complex cellular processes such as signaling and membrane trafficking. The yeast system offers many advantages for such studies, including the relative simplicity of its lipidome as compared to mammalian cells, the relatively high proportion of structural and regulatory genes of lipid metabolism which have been assigned and the excellent tools for molecular genetic analysis that yeast affords. The current state of application of lipidomic approaches in yeast and the advantages and disadvantages of yeast for such studies are discussed in this report.
Assuntos
Lipídeos/fisiologia , Leveduras/fisiologia , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Inositol/metabolismo , Membranas Intracelulares/química , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Metabolismo dos Lipídeos , Lipídeos de Membrana/química , Ácidos Fosfatídicos/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Although antioxidants are used to treat an overdose of the analgaesic/antipyretic drug APAP (acetaminophen), roles of antioxidant enzymes in APAP-induced hepatotoxicity remain controversial. Our objective was to determine impacts of knockout of SOD1 (superoxide dismutase; Cu,Zn-SOD) alone or in combination with selenium-dependent GPX1 (glutathione peroxidase-1) on APAP-induced hepatotoxicity. All SOD1-null (SOD1-/-) and SOD1- and GPX1-double-knockout mice survived an intraperitoneal injection of 600 mg of APAP per kg of body mass, whereas 75% of WT (wild-type) and GPX1-null mice died within 20 h. Survival time of SOD1-/- mice injected with 1200 mg of APAP per kg of body mass was longer than that of the WT mice (934 compared with 315 min, P<0.05). The APAP-treated SOD1-/- mice had less (P<0.05) plasma ALT (alanine aminotransferase) activity increase and attenuated (P<0.05) hepatic glutathione depletion than the WT mice. The protection conferred by SOD1 deletion was associated with a block of the APAP-mediated hepatic protein nitration and a 50% reduction (P<0.05) in activity of a key APAP metabolism enzyme CYP2E1 (cytochrome P450 2E1) in liver. The SOD1 deletion also caused moderate shifts in the APAP metabolism profiles. In conclusion, deletion of SOD1 alone or in combination with GPX1 greatly enhanced mouse resistance to APAP overdose. Our results suggest a possible pro-oxidant role for the physiological level of SOD1 activity in APAP-mediated hepatotoxicity.
Assuntos
Acetaminofen/toxicidade , Glutationa Peroxidase/fisiologia , Falência Hepática/induzido quimicamente , Superóxido Dismutase/deficiência , Acetaminofen/farmacocinética , Alanina Transaminase/biossíntese , Animais , Benzoquinonas/metabolismo , Benzoquinonas/toxicidade , Biotransformação , Peso Corporal , Citocromo P-450 CYP2E1/fisiologia , Inibidores do Citocromo P-450 CYP2E1 , Resistência a Medicamentos , Indução Enzimática/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Peroxidase/deficiência , Glutationa Peroxidase/genética , Iminas/metabolismo , Iminas/toxicidade , Falência Hepática/enzimologia , Falência Hepática/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Estresse Oxidativo , Ácido Peroxinitroso/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/fisiologia , Tirosina/análogos & derivados , Tirosina/biossíntese , Glutationa Peroxidase GPX1RESUMO
OBJECTIVE: Resveratrol, a constituent found in grapes and various other plants, has been shown to have chemo-preventive activity against cancer, and specifically demonstrated to induce apoptosis by p53-dependent pathways in murine cells. The goal of this research was to identify the role of p53-dependent or p53-independent pathways in the induction of apoptosis in human breast cancer cells by this natural product. DESIGN: A number of human breast cancer cell lines, as well as a control of a wild-type line (astrocytoma N 1321N1), were investigated for induction of apoptosis by resveratrol using both microscopic evaluation and DNA fragmentation assays. Concurrently, we established the p53 gene status (wild-type or mutant) of each cell line by Western blot using p53-specific antibody. RESULTS: Apoptosis induced by resveratrol was found to occur only in breast cancer cells expressing wild-type p53 but not in mutant p53-expressing cells. CONCLUSIONS: We therefore conclude that the natural product, resveratrol, induces apoptosis in breast cancer cells via p53-dependent pathways.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Estilbenos/farmacologia , Proteína Supressora de Tumor p53/efeitos dos fármacos , Antioxidantes/farmacologia , Astrocitoma/tratamento farmacológico , Astrocitoma/metabolismo , Western Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
We have previously shown a more potent impact of knockout of Cu,Zn-superoxide dismutase (SOD1) than that of Se-dependent glutathione peroxidase-1 (GPX1) on murine hepatotoxicity induced by an intraperitoneal (ip) injection of a high dose of acetaminophen (APAP, 600 mg/kg). The objective of this experiment was to compare the temporal impacts of knockouts of GPX1 and SOD1 alone or together on mouse susceptibility to an injection of a low dose of APAP (300 mg/kg). The APAP-mediated rises in plasma alanine aminotransferase activity and nitrate/nitrite concentrations, hepatic GSH depletion, and hepatic protein nitration at 5 and (or) 24 h were nearly abolished (P < 0.05) in SOD1-/- or GPX1 and SOD1 double-knockout (DKO) mice, while GPX1-/- mice exerted only moderate or no change compared with the WT. Despite an increased (P < 0.05) APAP-N-acetylcysteine and decreased APAP-glucuronide (P < 0.05) relative to the total APAP metabolites in urine collected for 24 h after the APAP injection, the SOD1-/- mice displayed no shift in urinary APAP-cysteine compared with the WT mice. Knockout of SOD1 prevented the APAP-induced hepatic GPX inactivation (P < 0.05), whereas knockout of GPX1 aggravated the APAP-induced hepatic SOD activity loss (P < 0.05). However, the APAP-mediated activity changes of these enzymes in liver accompanied no protein alterations. In conclusion, knockout of GPX1 or SOD1 exerted differential impact on mouse susceptibility to this low dose of APAP, but neither shifted urinary APAP-cysteine formation.
Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Antioxidantes , Doença Hepática Induzida por Substâncias e Drogas/sangue , Glutationa Peroxidase , Superóxido Dismutase , Acetaminofen/farmacologia , Analgésicos não Narcóticos/farmacologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Camundongos , Camundongos Knockout , Nitratos/sangue , Nitritos/sangue , Superóxido Dismutase-1 , Transaminases/sangue , Glutationa Peroxidase GPX1RESUMO
The addition of inositol to actively growing yeast cultures causes a rapid increase in the rate of synthesis of phosphatidylinositol and, simultaneously, triggers changes in the expression of hundreds of genes. We now demonstrate that the addition of inositol to yeast cells growing in the presence of choline leads to a dramatic reprogramming of cellular lipid synthesis and turnover. The response to inositol includes a 5-6-fold increase in cellular phosphatidylinositol content within a period of 30 min. The increase in phosphatidylinositol content appears to be dependent upon fatty acid synthesis. Phosphatidylcholine turnover increased rapidly following inositol addition, a response that requires the participation of Nte1p, an endoplasmic reticulum-localized phospholipase B. Mass spectrometry revealed that the acyl species composition of phosphatidylinositol is relatively constant regardless of supplementation with inositol or choline, whereas phosphatidylcholine acyl species composition is influenced by both inositol and choline. In medium containing inositol, but lacking choline, high levels of dimyristoylphosphatidylcholine were detected. Within 60 min following the addition of inositol, dimyristoylphosphatidylcholine levels had decreased from approximately 40% of total phosphatidylcholine to a basal level of less than 5%. nte1Delta cells grown in the absence of inositol and in the presence of choline exhibited lower levels of dimyristoylphosphatidylcholine than wild type cells grown under these same conditions, but these levels remained largely constant after the addition of inositol. These results are discussed in relationship to transcriptional regulation known to be linked to lipid metabolism in yeast.