RESUMEN
G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.
Asunto(s)
G-Cuádruplex , Regulación del Desarrollo de la Expresión Génica , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , ADN de Cadena Simple , Embrión no Mamífero/metabolismo , Humanos , Ratones , Regiones Promotoras Genéticas , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
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.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/genética , Diferenciación Celular/efectos de los fármacos , Neuronas/metabolismo , Fosfolípidos/biosíntesis , Animales , Proteína beta Potenciadora de Unión a CCAAT/biosíntesis , Proliferación Celular , Colina Quinasa/biosíntesis , Colina Quinasa/metabolismo , Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/metabolismo , Humanos , Ratones , Neuritas/metabolismo , Neuronas/citología , Fosfatidilcolinas/metabolismo , Fosfolípidos/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 µM) for 48 h exhibited a dose-response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Glutatión Transferasa/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteína de Unión a CREB/metabolismo , Células CACO-2 , Colforsina/farmacología , Dinitroclorobenceno/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Factor de Transcripción AP-1/metabolismoRESUMEN
In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.
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Bufo arenarum/embriología , Colina Quinasa/metabolismo , Citidililtransferasa de Colina-Fosfato/metabolismo , Embrión no Mamífero/enzimología , Fosfatidilcolinas/metabolismo , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Animales , Bufo arenarum/metabolismo , Colina Quinasa/genética , Citidililtransferasa de Colina-Fosfato/genética , Citosol/enzimología , Femenino , Masculino , Óvulo/enzimología , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/metabolismoRESUMEN
Antimicrobial resistance is a global public health threat. Metallo-ß-lactamases (MBLs) inactivate ß-lactam antibiotics, including carbapenems, are disseminating among Gram-negative bacteria, and lack clinically useful inhibitors. The evolving bisthiazolidine (BTZ) scaffold inhibits all three MBL subclasses (B1-B3). We report design, synthesis, and evaluation of BTZ analogues. Structure-activity relationships identified the BTZ thiol as essential, while carboxylate is replaceable, with its removal enhancing potency by facilitating hydrophobic interactions within the MBL active site. While the introduction of a flexible aromatic ring is neutral or detrimental for inhibition, a rigid (fused) ring generated nM benzobisheterocycle (BBH) inhibitors that potentiated carbapenems against MBL-producing strains. Crystallography of BBH:MBL complexes identified hydrophobic interactions as the basis of potency toward B1 MBLs. These data underscore BTZs as versatile, potent broad-spectrum MBL inhibitors (with activity extending to enzymes refractory to other inhibitors) and provide a rational approach to further improve the tricyclic BBH scaffold.
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Antibacterianos , Inhibidores de beta-Lactamasas , Inhibidores de beta-Lactamasas/farmacología , Inhibidores de beta-Lactamasas/química , Antibacterianos/farmacología , Antibacterianos/química , beta-Lactamasas/química , Carbapenémicos , Bacterias GramnegativasRESUMEN
Multidrug resistance-associated protein 3 (Mrp3; Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas multidrug resistance-associated protein 2 (Mrp2) is down-regulated. This study was undertaken to determine whether Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, noncholestatic dose of EE (5 mg/kg s.c.), and basal bile flow and the biliary excretion rate of bile salts and glutathione were measured 5 hours later. This treatment increased Mrp3 mRNA by 4-fold, detected by real-time polymerase chain reaction, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1-10 µM) for 5 hours exhibited a 3-fold increase in Mrp3 mRNA (10 µM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were incubated with an ER antagonist [7α,17ß-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI182/780), 1 µM], in addition to EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 minutes and expression of c-Jun, a well-known ER target gene, at 60 minutes, as detected by Western blotting of nuclear extracts. These increases were prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation and required participation of an ER, most likely ER-α, through its phosphorylation.
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Colestasis/metabolismo , Receptor alfa de Estrógeno/agonistas , Estrógenos/farmacología , Etinilestradiol/farmacología , Hígado/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/efectos de los fármacos , Animales , Bilis/metabolismo , Ácidos y Sales Biliares/metabolismo , Células Cultivadas , Colestasis/genética , Dactinomicina/farmacología , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Fulvestrant , Glutatión/metabolismo , Hígado/metabolismo , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosforilación , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Extracellular vesicles (EVs) are released by all cell types and are involved in intercellular communication. We evaluated if neural stem cells-derived EVs (NSC-EVs) regulate NSCs proliferation and differentiation under control and stress conditions. We found that NSC-EVs treatment increases cell proliferation and promotes neuronal differentiation and plasticity. The fact that nervous tissue poorly recovers after cellular damage, prump us to evaluate the effect of EVs supplementation under oxidative stress and inflammation. We demonstrate that NSC-EVs restore the proliferative potential of the NSCs affected by oxidative stress. In addition, we provide evidence that oxidative stress and inflammation induce neuronal differentiation. Interestingly, the aberrant cell phenotype induced by inflammation is restored by NSC-EVs treatment, suggesting that these vesicles ameliorate the damage burden in neurons and modulate neuronal plasticity. These results contribute to understand the role of the NSCs-derived EVs as key players for brain tissue generation and regeneration and open new pathways to the development of therapies.
RESUMEN
Neuroblastoma, the most common extracranial solid tumor occurring in childhood, originates from the aberrant proliferation of neural crest cells. Accordingly, the mechanism underling neuronal differentiation could provide new strategies for neuroblastoma treatment. It is well known that neurite outgrowth could be induced by Angiotensin II (Ang II) AT2 receptors; however, the signaling mechanism and its possible interaction with NGF (neural growth factor) receptors remain unclear. Here, we show that Ang II and CGP42112A (AT2 receptor agonist) promote neuronal differentiation by inducing neurite outgrowth and ßIII-tubulin expression in SH-SY5Y neuroblastoma cells. In addition, we demonstrate that treatment with PD123319 (AT2 receptor antagonist) reverts Ang II or CGP42112A-induced differentiation. By using specific pharmacological inhibitors we established that neurite outgrowth induced by CGP42112A requires the activation of MEK (mitogen-activated protein kinase kinase), SphK (sphingosine kinase) and c-Src but not PI3K (phosphatidylinositol 3-kinase). Certainly, CGP42112A stimulated a rapid and transient (30 s, 1 min) phosphorylation of c-Src at residue Y416 (indicative of activation), following by a Src deactivation as indicated by phosphorylation of Y527. Moreover, inhibition of the NGF receptor tyrosine kinase A (TrkA) reduced neurite outgrowth induced by Ang II and CGP42112A. In summary, we demonstrated that AT2 receptor-stimulated neurite outgrowth in SH-SY5Y cells involves the induction of MEK, SphK and c-Src and suggests a possible transactivation of TrkA. In that regard, AT2 signaling pathway is a key player in neuronal differentiation and might be a potential target for therapeutic treatments.
RESUMEN
[This corrects the article DOI: 10.1039/D0SC05172A.].
RESUMEN
Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes that are dependent on membrane biosynthesis. Thus, the production of phosphatidylcholine (PtdCho), the major membrane phospholipid, should be stimulated during neuronal differentiation. We demonstrate that during retinoic acid (RA)-induced differentiation of Neuro-2a cells, PtdCho synthesis was promoted by an ordered and sequential activation of choline kinase alpha (CK(alpha)) and choline cytidylyltransferase alpha (CCT(alpha)). Early after RA stimulation, the increase in PtdCho synthesis is mainly governed by the biochemical activation of CCT(alpha). Later, the transcription of CK(alpha)- and CCT(alpha)-encoding genes was induced. Both PtdCho biosynthesis and neuronal differentiation are dependent on ERK activation. A novel mechanism is proposed by which PtdCho biosynthesis is coordinated during neuronal differentiation. Enforced expression of either CK(alpha) or CCTalpha increased the rate of synthesis and the amount of PtdCho, and these cells initiated differentiation without RA stimulation, as evidenced by cell morphology and the expression of genes associated with neuritogenesis. The differentiation resulting from enforced expression of CCT(alpha) or CK(alpha) was dependent on persistent ERK activation. These results indicate that elevated PtdCho synthesis could mimic the RA signals and thus determine neuronal cell fate. Moreover, they could explain the key role that PtdCho plays during neuronal regeneration.
Asunto(s)
Neuronas/citología , Neuronas/metabolismo , Fosfatidilcolinas/biosíntesis , Animales , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Colina Quinasa/genética , Citidililtransferasa de Colina-Fosfato/genética , Técnica del Anticuerpo Fluorescente , Ratones , Neuronas/efectos de los fármacos , Neuronas/enzimología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilcolinas/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tretinoina/farmacologíaRESUMEN
CTP:phosphocholine cytidylyltransferase alpha (CCTalpha) is a key enzyme for phosphatidylcholine biosynthesis in mammalian cells. This enzyme plays an essential role in all processes that require membrane biosynthesis such as cell proliferation and viability. Thus, CCTalpha activity and expression fluctuate during the cell cycle to achieve PtdCho requirements. We demonstrated, for the first time, that CCTalpha is localized in the nucleus in cells transiting the S phase, whereas it is localized in the cytoplasm of G(0)-arrested cells, suggesting a specific role of nuclear CCTalpha during the S phase. We also investigated how E2F1 influences the regulation of the CCTalpha-promoter during the S phase; we demonstrated that E2F1 is necessary, but not sufficient, to activate CCTalpha expression when this factor is over-expressed. However, when E2F1 and Sp1 were over-expressed, the transcription from the CCTalpha-promoter reporter construct was super-activated. Transient transfection studies demonstrated that E2F1 could super-activate Sp1-dependent transcription in a promoter containing only the Sp1 binding sites "B" or "C", and that Sp1 could activate Sp1-dependent transcription in a promoter containing the E2F site, thus, further demonstrating a functional interaction of these factors. In conclusion, the present results allowed us to portray the clearest picture of the CCTalpha-gene expression in proliferating cells, and understand the mechanism by which cells coordinate cell cycle progression with the requirement for phosphatidylcholine.
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Citidililtransferasa de Colina-Fosfato/metabolismo , Factor de Transcripción E2F1/metabolismo , Fase S , Factor de Transcripción Sp1/metabolismo , Animales , Sitios de Unión , Citidililtransferasa de Colina-Fosfato/genética , Inmunoprecipitación de Cromatina , Citidina Trifosfato/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Regulación Enzimológica de la Expresión Génica , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C3H , Regiones Promotoras GenéticasRESUMEN
Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes, which are critically dependent on membrane biosynthesis, and therefore, on the expression and regulation of enzymes involved in phospholipid biosynthesis. During the last decade a great effort was made to clarify where membrane lipids are synthesized, how the newly synthesized membrane components reach the membrane and are inserted during neuritogenesis and to elucidate the mechanism by which the supply of new membrane components is coordinated with the demand for growth. Phosphatidylcholine is the principal and essential component for mammalian membranes. This review updates the mechanism by which phosphatidylcholine biosynthesis takes place and how it is coordinately regulated during neuronal differentiation.
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Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Neuritas/fisiología , Neuronas/metabolismo , Fosfatidilcolinas/biosíntesis , Fosfatidilcolinas/metabolismo , Animales , Colina Quinasa/metabolismo , Citidililtransferasa de Colina-Fosfato/metabolismo , Diacilglicerol Colinafosfotransferasa/metabolismo , Humanos , Mamíferos , Modelos Biológicos , Neuronas/citologíaRESUMEN
The balances between NSCs growth and differentiation, and between glial and neuronal differentiation play a key role in brain regeneration after any pathological conditions. It is well known that the nervous tissue shows a poor recovery after injury due to the factors present in the wounded microenvironment, particularly inflammatory factors, that prevent neuronal differentiation. Thus, it is essential to generate a favourable condition for NSCs and conduct them to differentiate towards functional neurons. Here, we show that neuroinflammation has no effect on NSCs proliferation but induces an aberrant neuronal differentiation that gives rise to dystrophic, non-functional neurons. This is perhaps the initial step of brain failure associated to many neurological disorders. Interestingly, we demonstrate that phosphatidylcholine (PtdCho)-enriched media enhances neuronal differentiation even under inflammatory stress by modifying the commitment of post-mitotic cells. The pro-neurogenic effect of PtdCho increases the population of healthy normal neurons. In addition, we provide evidences that this phospholipid ameliorates the damage of neurons and, in consequence, modulates neuronal plasticity. These results contribute to our understanding of NSCs behaviour under inflammatory conditions, opening up new venues to improve neurogenic capacity in the brain.
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Plasticidad de la Célula/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Fosfatidilcolinas/farmacología , Sinapsis/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Fenotipo , Células RAW 264.7 , Sinapsis/metabolismo , Sinapsis/patologíaRESUMEN
Infections caused by multidrug resistant (MDR) bacteria are a major public health threat. Carbapenems are among the most potent antimicrobial agents that are commercially available to treat MDR bacteria. Bacterial production of carbapenem-hydrolysing metallo-ß-lactamases (MBLs) challenges their safety and efficacy, with subclass B1 MBLs hydrolysing almost all ß-lactam antibiotics. MBL inhibitors would fulfil an urgent clinical need by prolonging the lifetime of these life-saving drugs. Here we report the synthesis and activity of a series of 2-mercaptomethyl-thiazolidines (MMTZs), designed to replicate MBL interactions with reaction intermediates or hydrolysis products. MMTZs are potent competitive inhibitors of B1 MBLs in vitro (e.g., K i = 0.44 µM vs. NDM-1). Crystal structures of MMTZ complexes reveal similar binding patterns to the most clinically important B1 MBLs (NDM-1, VIM-2 and IMP-1), contrasting with previously studied thiol-based MBL inhibitors, such as bisthiazolidines (BTZs) or captopril stereoisomers, which exhibit lower, more variable potencies and multiple binding modes. MMTZ binding involves thiol coordination to the Zn(ii) site and extensive hydrophobic interactions, burying the inhibitor more deeply within the active site than d/l-captopril. Unexpectedly, MMTZ binding features a thioether-π interaction with a conserved active-site aromatic residue, consistent with their equipotent inhibition and similar binding to multiple MBLs. MMTZs penetrate multiple Enterobacterales, inhibit NDM-1 in situ, and restore carbapenem potency against clinical isolates expressing B1 MBLs. Based on their inhibitory profile and lack of eukaryotic cell toxicity, MMTZs represent a promising scaffold for MBL inhibitor development. These results also suggest sulphur-π interactions can be exploited for general ligand design in medicinal chemistry.
RESUMEN
Metallo-ß-lactamase (MBL) production in Gram-negative bacteria is an important contributor to ß-lactam antibiotic resistance. Combining ß-lactams with ß-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of "cross-class" MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, Ki 0.16 µM) and potentiate ß-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono- or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.
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Inhibidores de beta-Lactamasas , beta-Lactamasas , Antibacterianos/farmacología , Tiazolidinas , Inhibidores de beta-Lactamasas/farmacología , beta-LactamasRESUMEN
Phosphatidylcholine biosynthesis in animal cells is primarily regulated by the rapid translocation of CTP:phosphocholine cytidylyltransferase alpha between a soluble form that is inactive and a membrane-associated form that is activated. Until less than 10 years ago there was no information on the transcriptional regulation of phosphatidylcholine biosynthesis. Research has identified the transcription factors Sp1, Rb, TEF4, Ets-1 and E2F as enhancing the expression of the cytidylyltransferase and Net as a factor that represses cytidylyltransferase expression. Key transcription factors involved in cholesterol or fatty acid metabolism (SREBPs, LXRs, PPARs) do not have a major role in transcriptional regulation of the cytidylyltransferase. Rather than being linked to cholesterol or energy metabolism, regulation of the cytidylyltransferase is linked to the cell cycle, cell growth and differentiation. Transcriptional regulation of phospholipid biosynthesis is more elegantly understood in yeast and involves responses to inositol, choline and zinc in the culture medium.
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Fosfatidilcolinas/biosíntesis , Transcripción Genética , Animales , Secuencia de Bases , Ciclo Celular/fisiología , Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/fisiología , Biología Computacional , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Fosfatidilcolinas/genética , Fosfolípidos/genética , Proteína Proto-Oncogénica c-ets-1/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción Sp1/fisiologíaRESUMEN
CTP:phosphocholine cytidylyltransferase (CCT) is a key regulatory enzyme in phosphatidylcholine (PtdCho) biosynthesis by the Kennedy pathway. In mammals, there are two genes that encode the enzyme isoforms that catalyze this reaction: Pcyt1a for CCTalpha and Pcyt1b for CCTbeta. In mouse tissues two different CCTbeta variants named CCTbeta2 and CCTbeta3 have been identified. Although little is known about Pcyt1b gene expression, recent data from cell lines propose a distinct role for CCTbeta2 in neuronal differentiation. Also, gonadal dysfunction in the CCTbeta2 knockout mouse suggests a role for this protein in ovary maturation and the maintenance of sperm production. This work defines and characterizes two alternative promoters that drive the expression of the two murine CCTbeta isoforms. The promoter activities were measured in Neuro-2a (mouse neuroblastoma), TM4 (mouse Sertoli) and C3H10T1/2 (mouse embryo fibroblast) cell lines. The transcriptional start points of each transcript and the promoter regions essential for the expression of each isoform were determined. Analysis of the CCTbeta2 promoter sequence suggested the transcription factor AP-1 as a potential regulator of CCTbeta2 expression in neuronal cells. However, CCTbeta3 was not detected in this cell line suggesting a different role or regulation. The activities of alternative promoters provide for greater flexibility in the control of CCTbeta isoform expression.
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Citidililtransferasa de Colina-Fosfato/genética , Isoenzimas/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Encéfalo/enzimología , Línea Celular , Citidililtransferasa de Colina-Fosfato/metabolismo , Biología Computacional , Eliminación de Gen , Isoenzimas/metabolismo , Ratones , Datos de Secuencia MolecularRESUMEN
Microorganisms, plants and animals regulate the synthesis of unsaturated fatty acids (UFAs) during changing environmental conditions as well as in response to nutrients. Unsaturation of fatty acid chains has important structural roles in cell membranes: a proper ratio of saturated to UFAs contributes to membrane fluidity. Alterations in this ratio have been implicated in various disease states including cardiovascular diseases, immune disorders, cancer and obesity. They are also the major components of triglycerides and intermediates in the synthesis of biologically active molecules such as eicosanoids, which mediates fever, inflammation and neurotransmission. UFAs homeostasis in many organisms is achieved by feedback regulation of fatty acid desaturases gene transcription. Here, we review recently discovered components and mechanisms of the regulatory machinery governing the transcription of fatty acid desaturases in bacteria, yeast and animals.
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Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Insaturados/fisiología , Transducción de Señal/fisiología , Animales , Bacillus subtilis/metabolismo , Regulación de la Expresión Génica , Humanos , Insulina/fisiología , Leptina/fisiología , Fluidez de la Membrana/fisiología , Receptores Activados del Proliferador del Peroxisoma/fisiología , Pseudomonas aeruginosa/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/fisiología , Synechocystis/metabolismoRESUMEN
The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells.
Asunto(s)
Colina Quinasa/genética , Proteínas F-Box/metabolismo , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/metabolismo , Neuroblastoma/genética , Tretinoina/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Colina Quinasa/metabolismo , Epigénesis Genética , Proteínas F-Box/genética , Estudios de Seguimiento , Técnicas de Silenciamiento del Gen , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Ratones , Células-Madre Neurales/fisiología , Neuroblastoma/mortalidad , Neuroblastoma/patología , Pronóstico , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/metabolismo , Regulación hacia ArribaRESUMEN
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