RESUMO
Lysophosphatidic acid (LPA) is an abundant bioactive phospholipid, with multiple functions both in development and in pathological conditions. Here, we review the literature about the differential signaling of LPA through its specific receptors, which makes this lipid a versatile signaling molecule. This differential signaling is important for understanding how this molecule can have such diverse effects during central nervous system development and angiogenesis; and also, how it can act as a powerful mediator of pathological conditions, such as neuropathic pain, neurodegenerative diseases, and cancer progression. Ultimately, we review the preclinical and clinical uses of Autotaxin, LPA, and its receptors as therapeutic targets, approaching the most recent data of promising molecules modulating both LPA production and signaling. This review aims to summarize the most update knowledge about the mechanisms of LPA production and signaling in order to understand its biological functions in the central nervous system both in health and disease.
Assuntos
Lisofosfolipídeos/genética , Neovascularização Patológica/genética , Fosfolipídeos/genética , Humanos , Lisofosfolipídeos/metabolismo , Terapia de Alvo Molecular , Neovascularização Patológica/tratamento farmacológico , Fosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/uso terapêutico , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/uso terapêutico , Transdução de Sinais/genéticaRESUMO
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
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
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
Intestinal fatty acid-binding protein (IFABP) is highly expressed in the intestinal epithelium and it belongs to the family of soluble lipid binding proteins. These proteins are thought to participate in most aspects of the biology of lipids, regulating its availability for specific metabolic pathways, targeting and vectorial trafficking of lipids to specific subcellular compartments. The present study is based on the ability of IFABP to interact with phospholipid membranes, and we characterized its immersion into the bilayer's hydrophobic central region occupied by the acyl-chains. We constructed a series of Trp-mutants of IFABP to selectively probe the interaction of different regions of the protein, particularly the elements forming the portal domain that is proposed to regulate the exit and entry of ligands to/from the binding cavity. We employed several fluorescent techniques based on selective quenching induced by soluble or membrane confined agents. The results indicate that the portal region of IFABP penetrates deeply into the phospholipid bilayer, especially when CL-containing vesicles are employed. The orientation of the protein and the degree of penetration were highly dependent on the lipid composition, the superficial net charge and the ionic strength of the medium. These results may be relevant to understand the mechanism of ligand transfer and the specificity responsible for the unique functions of each member of the FABP family.
Assuntos
Membrana Celular/química , Proteínas de Ligação a Ácido Graxo/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Substituição de Aminoácidos , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Bicamadas Lipídicas/metabolismo , Mutação de Sentido Incorreto , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Estrutura Terciária de Proteína , RatosRESUMO
A new haloalkaliphilic archaeon, strain B4(T), was isolated from the former lake Texcoco in Mexico. The cells were Gram-negative, pleomorphic-shaped, pink to red pigmented and aerobic. Strain B4(T) required at least 2.5 M NaCl for growth, with optimum growth at 3.4 M NaCl. It was able to grow over a pH range of 7.5-10.0 and temperature of 25-50 °C, with optimal growth at pH 9 and 37 °C. Cells are lysed in hypotonic treatment with less than 1.3 M NaCl. The major polar lipids of strain B4(T) were phosphatidylglycerol and methyl-phosphatidylglycerophosphate. Phospholipids were detected, but not glycolipids. The nucleotide sequence of the 16S rRNA gene revealed that the strain B4(T) was phylogenetically related to members of the genus Natronorubrum. Sequence similarity with Natronorubrum tibetense was 96.28 %, with Natronorubrum sulfidifaciens 95.06 % and Natronorubrum sediminis 94.98 %. The G+C content of the DNA was 63.3 mol%. The name of Natronorubrum texcoconense sp. nov. is proposed. The type strain is B4(T) (=CECT 8067(T) = JCM 17497(T)).
Assuntos
Halobacteriaceae/classificação , Lagos/microbiologia , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Sequência de Bases , DNA Arqueal/genética , Genes de RNAr , Halobacteriaceae/genética , Halobacteriaceae/isolamento & purificação , Concentração de Íons de Hidrogênio , México , Dados de Sequência Molecular , Fosfolipídeos/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Cloreto de Sódio , TemperaturaRESUMO
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
Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially for the control of economical important insect pests. These are pore-forming toxins that interact with different receptors in the insect gut, forming pores in the apical membrane causing cell burst and insect death. Elucidation of the structure of the membrane-inserted toxin is important to fully understand its mechanism of action. One hypothesis proposed that the hairpin of α-helices 4-5 of domain I inserts into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane surface in an umbrella-like conformation. However, a second hypothesis proposed that the three domains of the Cry toxin insert into the bilayer without major conformational changes. In this work we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and labeled them with different fluorescent probes that have different responses to solvent polarity. Different soluble quenchers as well as a membrane-bound quencher were used to compare the properties of the soluble and brush border membrane-inserted forms of Cry1Ab toxin. The fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin remain in the surface of the membrane and only a discrete region of domain I is inserted into the lipid bilayer, supporting the umbrella model of toxin insertion.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/química , Membrana Celular/química , Endotoxinas/química , Proteínas Hemolisinas/química , Bicamadas Lipídicas/química , Modelos Químicos , Fosfolipídeos/química , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Larva/microbiologia , Bicamadas Lipídicas/metabolismo , Manduca/microbiologia , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Estrutura Terciária de ProteínaRESUMO
Bacteria stringently regulate the synthesis of their membrane phospholipids, but the responsible regulatory mechanisms are incompletely understood. Bacillus subtilis FabF, the target of the mycotoxin cerulenin, catalyses the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. Here we show that B. subtilis strains containing the fabF1 allele, which codes for the cerulenin-insensitive protein FabF[I108F], overexpressed several genes involved in fatty acid and phospholipid biosynthesis (the fap regulon) and had significantly elevated levels of malonyl-CoA. These results pinpointed FabF[I108F] as responsible for the increased malonyl-CoA production, which in turn acts as an inducer of the fap regulon by impairing the binding of the FapR repressor to its DNA targets. Synthesis of acyl-ACPs by a cell-free fatty acid system prepared from fabF1 cells showed the accumulation of short- and medium-chain acyl-ACPs. These results indicate that the acyl-ACP chain length acceptance of FabF[I108F] is biased towards shorter acyl-ACPs. We also provide evidence that upregulation of FabF[I108F] is essential for survival and for resistance to cerulenin of fabF1 cells. These findings indicate that malonyl-CoA is a key molecule to monitor lipid metabolism functioning and trigger appropriate genetic and biochemical adjustments to relieve dysfunctions of this essential metabolic pathway.