RESUMO
Glioblastoma multiforme is the most aggressive type of tumor of the CNS with an overall survival rate of approximately one year. Since this rate has not changed significantly over the last 20 years, the development of new therapeutic strategies for the treatment of these tumors is peremptory. The over-expression of the proto-oncogene c-Fos has been observed in several CNS tumors including glioblastoma multiforme and is usually associated with a poor prognosis. Besides its genomic activity as an AP-1 transcription factor, this protein can also activate phospholipid synthesis by a direct interaction with key enzymes of their metabolic pathways. Given that the amino-terminal portion of c-Fos (c-Fos-NA: amino acids 1-138) associates to but does not activate phospholipid synthesizing enzymes, we evaluated if c-Fos-NA or some shorter derivatives are capable of acting as dominant-negative peptides of the activating capacity of c-Fos. The over-expression or the exogenous administration of c-Fos-NA to cultured T98G cells hampers the interaction between c-Fos and PI4K2A, an enzyme activated by c-Fos. Moreover, it was observed a decrease in tumor cell proliferation rates in vitro and a reduction in tumor growth in vivo when a U87-MG-generated xenograft on nude mice is intratumorally treated with recombinant c-Fos-NA. Importantly, a smaller peptide of 92 amino acids derived from c-Fos-NA retains the capacity to interfere with tumor proliferation in vitro and in vivo. Taken together, these results support the use of the N-terminal portion of c-Fos, or shorter derivatives as a novel therapeutic strategy for the treatment of glioblastoma multiforme.
Assuntos
Proliferação de Células , Glioblastoma/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfolipídeos/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Antígenos de Histocompatibilidade Menor/genética , Fosfolipídeos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.
Assuntos
Bacillus subtilis/metabolismo , Ácidos Graxos/biossíntese , Fosfolipídeos/biossíntese , Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Ácidos Graxos/metabolismo , Lipogênese , Fosfolipídeos/metabolismo , RegulonRESUMO
PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.
Assuntos
Proteínas de Bactérias/genética , Redes e Vias Metabólicas/genética , Ácidos Fosfatídicos/metabolismo , Fosfolipídeos/biossíntese , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Catálise , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Lipogênese/genética , Ácidos Fosfatídicos/genética , Fosfolipídeos/genéticaRESUMO
Macrophage classical M1 activation via TLR4 triggers a variety of responses to achieve the elimination of foreign pathogens. During this process, there is also an increase in lipid droplets which contain large quantities of triacylglycerol (TAG) and phospholipid (PL). The functional consequences of this increment in lipid mass are poorly understood. Here, we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Using bone marrow-derived macrophages (BMDMs) treated with Kdo2-lipid A, we showed that glycerolipid synthesis is induced during macrophage activation. GPAT4 protein level and GPAT3/GPAT4 enzymatic activity increase during this process, and these two isoforms were required for the accumulation of cell TAG and PL. The phagocytic capacity of Gpat3-/- and Gpat4-/- BMDM was impaired. Additionally, inhibiting fatty acid ß-oxidation reduced phagocytosis only partially, suggesting that lipid accumulation is not necessary for the energy requirements for phagocytosis. Finally, Gpat4-/- BMDM expressed and released more pro-inflammatory cytokines and chemokines after macrophage activation, suggesting a role for GPAT4 in suppressing inflammatory responses. Together, these results provide evidence that glycerolipid synthesis directed by GPAT4 is important for the attenuation of the inflammatory response in activated macrophages.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Lipogênese , Macrófagos/enzimologia , Fosfolipídeos/biossíntese , Triglicerídeos/biossíntese , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Animais , Glicerol-3-Fosfato O-Aciltransferase/genética , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Ativação de Macrófagos/genética , Macrófagos/patologia , Camundongos , Camundongos Knockout , Fosfolipídeos/genética , Triglicerídeos/genéticaRESUMO
PlsX is a central enzyme of phospholipid synthesis in bacteria, converting acyl-ACP to acyl-phosphate on the pathway to phosphatidic acid formation. PlsX has received attention because it plays a key role in the coordination of fatty acid and phospholipid synthesis. Recently, PlsX was also suggested to coordinate membrane synthesis with cell division in Bacillus subtilis. Here, we have re-investigated the cell biology of PlsX and determined that the enzyme is uniformly distributed on the membrane of most cells, but occasionally appears as membrane foci as well. Foci and homogenous patterns seem freely interconvertible but the prevalence of the uniform staining suggests that PlsX does not need to localize to specific sites to function correctly. We also investigated the relationship between PlsX and the divisome. In contrast to previous observations, PlsX's foci showed no obvious periodicity of localization and did not colocalize with the divisome. Furthermore, depletion of PlsX did not affect cell division if phospholipid synthesis is maintained by an alternative enzyme. These results suggest that coordination between division and membrane synthesis may not require physical or functional interactions between the divisome and phospholipid synthesis enzymes.
Assuntos
Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Divisão Celular , Fosfolipídeos/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Ácidos Graxos/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/metabolismoRESUMO
Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-ß (C/EBPß) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPß expression. Elevated levels of C/EBPß bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Neurônios/metabolismo , Fosfolipídeos/biossíntese , Animais , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proliferação de Células , Colina Quinase/biossíntese , Colina Quinase/metabolismo , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Humanos , Camundongos , Neuritos/metabolismo , Neurônios/citologia , Fosfatidilcolinas/metabolismo , Fosfolipídeos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
Lipid synthesis is a complex process regulated at multiple levels. Here, we will discuss nongenomic regulatory mechanisms, particularly the activation and/or recruitment of key enzymes to membranes. The phospholipid synthesis enzymes Lipin and CTP:phosphocholine cytidylyltransferase are taken as examples of these mechanisms that are mediated by posttranslational modifications or by an intrinsic property of the enzyme that senses lipid composition. In addition, special emphasis will be put on another relevant non genomic lipid synthesis regulation mechanism that is dependent on c-Fos, a protein that has deserved less attention so far. This latter regulatory mechanism is emerging as an important determinant for processes that require high rates of lipid synthesis such as those of growth and proliferation.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Fosfolipídeos/biossíntese , Processamento de Proteína Pós-Traducional , Membrana Celular/enzimologia , Proliferação de Células , Humanos , Lipídeos/biossíntese , Compostos Orgânicos/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
BACKGROUND: StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (-26.4% and -41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and (3)H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin ß-subunit (ßhCG) protein synthesis and secretion as well as ßhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7. CONCLUSIONS/SIGNIFICANCE: Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Transporte/genética , Diferenciação Celular/genética , Movimento Celular/genética , Coriocarcinoma/genética , Coriocarcinoma/patologia , Técnicas de Silenciamento de Genes , Proteínas de Neoplasias/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Biomarcadores/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Regulação Neoplásica da Expressão Gênica , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Inativação Gênica , Células Gigantes/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Fosfolipídeos/biossíntese , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Regulação para Cima/genéticaRESUMO
Some 20 years ago c-Fos was identified as a member of the AP-1 family of inducible transcription factors (Angel and Karin in Biochim Biophys Acta 1072:129-157, 1991). More recently, an additional activity was described for this protein: it associates to the endoplasmic reticulum and activates the biosynthesis of phospholipids (Bussolino et al. in FASEB J 15:556-558, 2001), (Gil et al. in Mol Biol Cell 15:1881-1894, 2004), the quantitatively most important components of cellular membranes. This latter activity of c-Fos determines the rate of membrane genesis and consequently of growth in differentiating PC12 cells (Gil et al. in Mol Biol Cell 15:1881-1894, 2004). In addition, it has been shown that c-Fos is over-expressed both in PNS and CNS tumors (Silvestre et al. in PLoS One 5(3):e9544, 2010). Herein, it is shown that c-Fos-activated phospholipid synthesis is required to support membrane genesis during the exacerbated growth characteristic of brain tumor cells. Specifically blocking c-Fos-activated phospholipid synthesis significantly reduces proliferation of tumor cells in culture. Blocking c-Fos expression also prevents tumor progression in mice intra-cranially xeno-grafted human brain tumor cells. In NPcis mice, an animal model of the human disease Neurofibromatosis Type I (Cichowski and Jacks in Cell 104:593-604, 2001), animals spontaneously develop tumors of the PNS and the CNS, provided they express c-Fos (Silvestre et al. in PLoS One 5(3):e9544, 2010). Treatment of PNS tumors with an antisense oligonucleotide that specifically blocks c-Fos expression also blocks tumor growth in vivo. These results disclose cytoplasmic c-Fos as a new target for effectively controlling brain tumor growth.
Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias do Sistema Nervoso Central/patologia , Neoplasias do Sistema Nervoso Periférico/patologia , Fosfolipídeos/biossíntese , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Linhagem Celular Tumoral , Sistema Nervoso Central/metabolismo , Neoplasias do Sistema Nervoso Central/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Camundongos , Oligonucleotídeos Antissenso/metabolismo , Células PC12 , Neoplasias do Sistema Nervoso Periférico/metabolismo , RatosRESUMO
Our previous work showed that in T98G cells, a human glioblastoma multiforme-derived cell line, the association of c-Fos to the endoplasmic reticulum (ER) and consequently, the capacity of c-Fos to activate phospholipid synthesis, is regulated by the phosphorylation state of tyrosine (tyr) residues #10 and #30 of c-Fos. The small amount of c-Fos present in quiescent cells is tyr-phosphorylated, is dissociated from the ER membranes and does not activate phospholipid synthesis. However, on induction of the cell to re-enter growth, c-Fos expression is rapidly induced, it is found dephosphorylated, associated to ER membranes and activating phospholipid synthesis (Portal et al., 2007). Herein, using in vivo and in vitro experimental strategies, we show that the kinase c-Src is capable of phosphorylating tyr residues of c-Fos whereas the phosphatase TC45 T-cell protein-tyr phosphatase (TC-PTP) dephosphorylates them, thus enabling c-Fos/ER association and activation of phospholipid synthesis. Results also suggest that the regulation of the phosphorylation/dephosphorylation cycle of c-Fos occurs at the TC-PTP level: induction of cells to re-enter growth promotes the translocation of TC45 from a nuclear to a cytoplasmic location concomitant with its activation. Activated TC45 in its turn promotes dephosphorylation of pre-formed c-Fos, enabling cells to rapidly activate phospholipid synthesis to respond to its growth demands.
Assuntos
Fosfolipídeos/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/metabolismo , Tirosina/metabolismo , Animais , Proteína Tirosina Quinase CSK , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Humanos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Cinética , Camundongos , Células NIH 3T3 , Fosforilação , Transporte Proteico , Quinases da Família srcRESUMO
The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II ß was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.
Assuntos
Fosfatos de Fosfatidilinositol/biossíntese , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição AP-1/metabolismo , 1-Fosfatidilinositol 4-Quinase/biossíntese , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Diacilglicerol Colinofosfotransferase/biossíntese , Diacilglicerol Colinofosfotransferase/genética , Ativação Enzimática/fisiologia , Indução Enzimática/fisiologia , Camundongos , Células NIH 3T3 , Fosfatos de Fosfatidilinositol/genética , Fosfolipídeos/biossíntese , Fosfolipídeos/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , Fator de Transcrição AP-1/genéticaRESUMO
It has been demonstrated that c-Fos has, in addition to its well recognized AP-1 transcription factor activity, the capacity to associate to the endoplasmic reticulum and activate key enzymes involved in the synthesis of phospholipids required for membrane biogenesis during cell growth and neurite formation. Because membrane genesis requires the coordinated supply of all its integral membrane components, the question emerges as to whether c-Fos also activates the synthesis of glycolipids, another ubiquitous membrane component. We show that c-Fos activates the metabolic labeling of glycolipids in differentiating PC12 cells. Specifically, c-Fos activates the enzyme glucosylceramide synthase (GlcCerS), the product of which, GlcCer, is the first glycosylated intermediate in the pathway of synthesis of glycolipids. By contrast, the activities of GlcCer galactosyltransferase 1 and lactosylceramide sialyltransferase 1 are essentially unaffected by c-Fos. Co-immunoprecipitation experiments in cells co-transfected with c-Fos and a V5-tagged version of GlcCerS evidenced that both proteins participate in a physical association. c-Fos expression is tightly regulated by specific environmental cues. This strict regulation assures that lipid metabolism activation will occur as a response to cell requirements thus pointing to c-Fos as an important regulator of key membrane metabolisms in membrane biogenesis-demanding processes.
Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Glucosiltransferases/metabolismo , Glicolipídeos/biossíntese , Neuritos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Diferenciação Celular/fisiologia , Ativação Enzimática/fisiologia , Células PC12 , Fosfolipídeos/biossíntese , Ligação Proteica/fisiologia , Ratos , Fator de Transcrição AP-1/metabolismoRESUMO
De acordo com a Organização Mundial da Saúde, AIDS, malária e tuberculose são as três maiores doenças infectantes do mundo, atingindo principalmente crianças. Regiões paupérrimas e de clima tropical, como a África sub-saariana, são as mais atingidas. Este quadro agrava-se com a disseminação de cepas do Plasmodium falcíparum resistentes à cloroquina e multi-resistentes. Além disso, alguns fármacos utilizados na terapêutica da malária apresentam vários efeitos adversos, comprometendo o tratamento. Trata-se de um grande desafio e o seu enfrentamento requer estratégias. O desenvolvimento de novos quimioterápicos deve fundamentar-se em diferenças bioquímicas e morfológicas entre as células do hospedeiro e do parasita. A biossíntese de fosfolipídeos de membrana em parasitas do grupo Apicomplexa é de extrema importância para a maturação e a reprodução do parasita e constitui-se em bom alvo para novos antimaláricos, uma vez que é encontrada somente em parasitas. Hemácias infectadas têm sua absorção modificada em relação aos eritrócitos não-infectados, conferindo seletividade a substâncias como lipídeos. O trabalho em questão propõe a síntese de antimetabólitos da serina, visando à inibição das enzimas fosfatidilserina síntase e serina descarboxilase, fundamentais para a biossíntese de fosfolipídeos de membrana desses parasitas. Cinco derivados heterocíclicos da serine foram sintetizados: derivados diidroimidazólico, diidroxazólico, diidroxazínico, diidropirimidínico e diidrooxatiólico. Também, o transportador fosfolipídico com o ácido esteárico foi sintetizado. Os antimetabólitos serão acoplados a esse e outros fosfolípídeos, obtendo-se fármacos dirigidos específicos direcionados seletivamente a eritrócitos infectados...
Assuntos
Antimaláricos/síntese química , Antimaláricos/uso terapêutico , Antimetabólitos/síntese química , Fosfolipídeos/biossíntese , Técnicas In Vitro , Malária/epidemiologia , Malária/tratamento farmacológico , Plasmodium falciparum/genética , Serina/farmacocinética , Serina/síntese química , Química Farmacêutica , Cromatografia/métodos , Cromatografia , Relação Estrutura-AtividadeRESUMO
Bacterial cells stringently regulate the synthesis of their membrane phospholipids but the responsible mechanisms are incompletely understood. Recent biochemical, genetic and structural analyses have greatly expanded the knowledge of lipid metabolism in Gram-positive bacteria, revealing that these organisms use novel mechanisms to regulate this essential pathway. A remarkable progress was the identification of a new pathway for the initiation of phospholipid biosynthesis that uncovered a mechanism that coordinates fatty acid and phospholipid biosynthesis. Recent advances in structure determination of a global transcription factor have led to significant insights of the underlying complexities and functional elegance of membrane lipid homeostasis in Gram-positive bacteria.
Assuntos
Ácido Graxo Sintase Tipo II/metabolismo , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Positivas/enzimologia , Lipídeos de Membrana/biossíntese , Ácido Graxo Sintase Tipo II/genética , Bactérias Gram-Positivas/genética , Lipídeos de Membrana/química , Modelos Moleculares , Fosfolipídeos/biossíntese , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
c-Fos dephosphorylated on tyrosine (c-Fos), a component of the activator protein-1 (AP-1) family of transcription factors, is expressed at very low levels in resting cells. However, its expression is rapidly upregulated in cells undergoing G(0) to S phase transition leading to AP-1-dependent gene transcription responses. In addition, cytoplasmic c-Fos associates to the endoplasmic reticulum (ER) membranes and activates phospholipid synthesis during cell growth and differentiation. Herein, it is shown that in T98G cells, c-Fos/ER association and consequently phospholipid synthesis activation is regulated by the phosphorylated state of c-Fos tyrosine (tyr) residues. The small amount of c-Fos present in quiescent T98G cells is tyr-phosphorylated and not ER-membrane bound. In growing cells, it is dephosphorylated, associated to ER membranes and promotes phospholipid synthesis activation. Impairing tyr-dephosphorylation abrogates phospholipid synthesis activation and reduces proliferation rates to those of quiescent cells. Substitution of tyr residues 10, 30, 106 and 337 evidence tyr 10 and 30 as relevant for this regulatory phenomenon. It is concluded that phosphorylation of tyr residues 10 and 30 of c-Fos regulate the rate of synthesis of phospholipids by regulating c-Fos/ER association.
Assuntos
Retículo Endoplasmático/metabolismo , Fosfolipídeos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Tirosina/metabolismo , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Humanos , Fosfolipídeos/biossíntese , Fosforilação/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genéticaRESUMO
Azospirillum-plant association is accompanied by biochemical changes in roots which, in turn, promote plant-growth and tolerance to water stress. To shed light on the possible factors underlying these effects, roots from Azospirillum brasilense Sp245-inoculated Triticum aestivum seedlings growing in darkness under osmotic stress were analyzed for phospholipid (PL) composition, fatty acid (FA) distribution profiles and degree of unsaturation of the major PL classes. Azospirillum inoculation diminished ion leakage and increased 2,3,5-tripheniltetrazolium reducing ability in roots of well irrigated and water-stressed wheat seedlings. Total root PL content remained unaltered in all treatments. Six PL classes were detected, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) comprising over 80% of the total. While water stress increased PC content and diminished that of PE, none of these changes were observed either under Azospirillum inoculation alone or when both treatments were combined. The major FAs found in both PC and PE were 16:0, 18:0, 18:1, 18:2, and 18:3. Higher PC and lower PE unsaturation than in well irrigated controls were observed in roots from Azospirillum-inoculated, water-stressed seedlings. Azospirillum inoculation could contribute to protect wheat seedlings from water stress through changes in the FA distribution profiles of PC and PE major root phospholipids.
Assuntos
Azospirillum , Fosfolipídeos/biossíntese , Raízes de Plantas/metabolismo , Plântula/metabolismo , Triticum/metabolismo , Azospirillum/crescimento & desenvolvimento , Azospirillum/metabolismo , Desidratação/metabolismo , Raízes de Plantas/microbiologia , Plântula/microbiologia , Triticum/microbiologiaRESUMO
Saturated and monounsaturated fatty acids are the most abundant fatty acid species in mammalian organisms, and their distribution is regulated by stearoyl-CoA desaturase, the enzyme that converts saturated into monounsaturated fatty acids. A positive correlation between high monounsaturated fatty acid levels and neoplastic transformation has been reported, but little is still known about the regulation of stearoyl-CoA desaturase in cell proliferation and apoptosis, as well as in cancer development. Here we report that simian virus 40-transformed human lung fibroblasts bearing a knockdown of human stearoyl-CoA desaturase by stable antisense cDNA transfection (hSCDas cells) showed a considerable reduction in monounsaturated fatty acids, cholesterol, and phospholipid synthesis, compared with empty vector transfected-simian virus 40 cell line (control cells). hSCDas cells also exhibited high cellular levels of saturated free fatty acids and triacylglycerol. Interestingly, stearoyl-CoA desaturase-depleted cells exhibited a dramatic decrease in proliferation rate and abolition of anchorage-independent growth. Prolonged exposure to exogenous oleic acid did not reverse either the slower proliferation or loss of anchorage-independent growth of hSCDas cells, suggesting that endogenous synthesis of monounsaturated fatty acids is essential for rapid cell replication and invasiveness, two hallmarks of neoplastic transformation. Moreover, apoptosis was increased in hSCDas cells in a ceramide-independent manner. Finally, stearoyl-CoA desaturase-deficient cells were more sensitive to palmitic acid-induced apoptosis compared with control cells. Our data suggest that, by globally regulating lipid metabolism, stearoyl-CoA desaturase activity modulates cell proliferation and survival and emphasize the important role of endogenously synthesized monounsaturated fatty acids in sustaining the neoplastic phenotype of transformed cells.
Assuntos
Fibroblastos/citologia , Fibroblastos/enzimologia , Estearoil-CoA Dessaturase/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Radioisótopos de Carbono , Adesão Celular , Divisão Celular/fisiologia , Linhagem Celular Transformada , Sobrevivência Celular/fisiologia , Ceramidas/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Homeostase/fisiologia , Humanos , Levanogestrel/toxicidade , Pulmão/citologia , Fosfolipídeos/biossíntese , Fosfolipídeos/metabolismo , Ácidos Esteáricos/farmacocinética , Triglicerídeos/metabolismoRESUMO
We recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis plays an important role in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells [Cerbon and Lopez-Sanchez (2003) Biochem. J. 373, 917-924]. In yeast cells, IPC (inositol phosphoceramide) synthase catalyses the transfer of phosphoinositol from phosphatidylinositol to ceramide to form IPC and generates DAG. In the present study, we found that, during the G1 to S transition after N2-starvation, there was a significant increase in the synthesis of IPC accompanied by a progressive increase (up to 6-fold) in the level of DAG. The increased DAG levels coincided with decrements in ceramide and sphingoid base levels, conditions that are adequate for the activation of putative protein kinase C required for the G1 to S transition and proliferation of yeast cells. To separate the role of DAG generated during IPC synthesis from that originating from other sources, we utilized beta-chloroalanine and myriocin, inhibitors of serine:palmitoyl-CoA transferase, the first committed step in sphingolipid synthesis, to avoid accumulation of sphingolipid intermediates. When the synthesis of sphingolipids was inhibited, DAG accumulation was significantly decreased and the G1 to S transition was blocked; such blockage was avoided by metabolic complementation with phytosphingosine. The DAG/ceramide ratio was 0.27 and it changed to 2.0 during growth re-initiation, suggesting that the synthesis of phosphosphingolipids could act to switch growth arrest (increased ceramide) to a mitogenic signal (increased DAG), and that this signalling process is preserved in yeast and mammalian cells.
Assuntos
Ceramidas/metabolismo , Diglicerídeos/metabolismo , Fase G1 , Hexosiltransferases/metabolismo , Fase S , Saccharomyces cerevisiae/enzimologia , Proliferação de Células , Depsipeptídeos/farmacologia , Ácidos Graxos Monoinsaturados/farmacologia , Hexosiltransferases/antagonistas & inibidores , Fosfolipídeos/biossíntese , Proteína Quinase C/metabolismo , Transdução de Sinais , Esfingolipídeos/biossíntese , beta-Alanina/análogos & derivados , beta-Alanina/farmacologiaRESUMO
Retinal ganglion cells (RGCs) are major components of the vertebrate circadian system. They send information to the brain, synchronizing the entire organism to the light-dark cycles. We recently reported that chicken RGCs display daily variations in the biosynthesis of glycerophospholipids in constant darkness (DD). It was unclear whether this rhythmicity was driven by this population itself or by other retinal cells. Here we show that RGCs present circadian oscillations in the labeling of [32P]phospholipids both in vivo in constant light (LL) and in cultures of immunopurified embryonic cells. In vivo, there was greater [32P]orthophosphate incorporation into total phospholipids during the subjective day. Phosphatidylinositol (PI) was the most 32P-labeled lipid at all times examined, displaying maximal levels during the subjective day and dusk. In addition, a significant daily variation was found in the activity of distinct enzymes of the pathway of phospholipid biosynthesis and degradation, such as lysophospholipid acyltransferases (AT II), phosphatidate phosphohydrolase (PAP), and diacylglycerol lipase (DGL) in cell preparations obtained in DD, exhibiting differential but coordinated temporal profiles. Furthermore, cultures of immunopurified RGCs synchronized by medium exchange displayed a circadian fluctuation in the phospholipid labeling. The results demonstrate that chicken RGCs contain circadian oscillators capable of generating metabolic oscillations in the biosynthesis of phospholipids autonomously.
Assuntos
Ritmo Circadiano/fisiologia , Ativação Enzimática , Luz , Fosfolipídeos/biossíntese , Células Ganglionares da Retina/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Animais , Animais Recém-Nascidos , Relógios Biológicos , Células Cultivadas , Embrião de Galinha , Galinhas , Escuridão , Técnicas In Vitro , Lipase Lipoproteica/metabolismo , Fosfatidato Fosfatase/metabolismo , Fosfatidilinositóis/metabolismo , Fosfoproteínas/metabolismo , Isótopos de Fósforo/metabolismo , Fatores de TempoRESUMO
The mammalian circadian timing system is composed of countless cell oscillators distributed throughout the body and central pacemakers regulating temporal physiology and behavior. Peripheral clocks display circadian rhythms in gene expression both in vivo and in culture. We examined the biosynthesis of phospholipids as well as the expression of the clock gene period 1 (Per1) and its potential involvement in the regulation of the phospholipid metabolism in cultured quiescent NIH 3T3 cells synchronized by a 2 h serum shock. A 30 min pulse of radiolabeled precursor was given at phases ranging from 0.5 to 62 h after serum treatment. We observed a daily rhythm in the phospholipid labeling that persisted at least for two cycles, with levels significantly decreasing 29 and 58 h after treatment. Per1 expression exhibited a rapid and transient induction and a daily rhythmicity in antiphase to the lipid labeling. After Per1 expression knockdown, the rhythm of phospholipid labeling was lost. Furthermore, in cultures of CLOCK mutant fibroblasts--cells with a clock mechanism impairment--PER1 was equally expressed at all times examined and the phospholipid labeling did not oscillate. The results demonstrate that the biosynthesis of phospholipids oscillates daily in cultured fibroblasts by an endogenous clock mechanism involving Per1 expression.