RESUMO
We report first prototypes of responsive lanthanide(III) complexes that can be monitored independently in three complementary imaging modalities. Through the appropriate choice of lanthanide(III) cations, the same reactive ligand can be used to form complexes providing detection by (i) visible (Tb(3+)) and near-infrared (Yb(3+)) luminescence, (ii) PARACEST- (Tb(3+), Yb(3+)), or (iii) T1-weighted (Gd(3+)) MRI. The use of lanthanide(III) ions of different natures for these imaging modalities induces only a minor change in the structure of complexes that are therefore expected to have a single biodistribution and cytotoxicity.
Assuntos
Elementos da Série dos Lantanídeos/química , Medições Luminescentes/métodos , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Meios de Contraste/químicaRESUMO
Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme.
Assuntos
Ensaios Enzimáticos , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frutosefosfatos/análise , Frutosefosfatos/metabolismo , Glucosamina/análogos & derivados , Glucosamina/análise , Glucosamina/metabolismo , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/análise , Glucose-6-Fosfato/metabolismo , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Glutamina/análise , Glutamina/metabolismo , Cinética , Especificidade por SubstratoRESUMO
The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Modelos Moleculares , Multimerização Proteica , Regulação Alostérica , Estabilidade Enzimática , Proteínas de Escherichia coli/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Estrutura Quaternária de Proteína , Relação Estrutura-AtividadeRESUMO
Enzyme-responsive MRI-contrast agents containing a "self-immolative" benzylcarbamate moiety that links the MRI-reporter lanthanide complex to a specific enzyme substrate have been developed. The enzymatic cleavage initiates an electronic cascade reaction that leads to a structural change in the Ln(III) complex, with a concomitant response in its MRI-contrast-enhancing properties. We synthesized and investigated a series of Gd(3+) and Yb(3+) complexes, including those bearing a self-immolative arm and a sugar unit as selective substrates for ß-galactosidase; we synthesized complex LnL(1), its NH(2) amine derivatives formed after enzymatic cleavage, LnL(2), and two model compounds, LnL(3) and LnL(4). All of the Gd(3+) complexes synthesized have a single inner-sphere water molecule. The relaxivity change upon enzymatic cleavage is limited (3.68 vs. 3.15 mM(-1) s(-1) for complexes GdL(1) and GdL(2), respectively; 37 °C, 60 MHz), which prevents application of this system as an enzyme-responsive T(1) relaxation agent. Variable-temperature (17)O NMR spectroscopy and (1)H NMRD (nuclear magnetic relaxation dispersion) analysis were used to assess the parameters that determine proton relaxivity for the Gd(3+) complexes, including the water-exchange rate (k(ex)(298), varies in the range 1.5-3.9×10(6) s(-1)). Following the enzymatic reaction, the chelates contain an exocyclic amine that is not protonated at physiological pH, as deduced from pH-potentiometric measurements (log K(H)=5.12(±0.01) and 5.99(±0.01) for GdL(2) and GdL(3), respectively). The Yb(3+) analogues show a PARACEST effect after enzymatic cleavage that can be exploited for the specific detection of enzymatic activity. The proton-exchange rates were determined at various pH values for the amine derivatives by using the dependency of the CEST effect on concentration, saturation time, and saturation power. A concentration-independent analysis of the saturation-power-dependency data was also applied. All these different methods showed that the exchange rate of the amine protons of the Yb(III) complexes decreases with increasing pH value (for YbL(3), k(ex)=1300 s(-1) at pH 8.4 vs. 6000 s(-1) at pH 6.4), thereby resulting in a diminution of the observed CEST effect.
Assuntos
Meios de Contraste/química , Elementos da Série dos Lantanídeos/química , Imageamento por Ressonância Magnética/métodos , Compostos Organometálicos/química , Gadolínio/química , Espectroscopia de Ressonância Magnética , Modelos Químicos , Estrutura Molecular , Compostos Organometálicos/síntese química , Água/química , Itérbio/químicaRESUMO
Streptomyces cattleya, a producer of the antibiotics thienamycin and cephamycin C, is one of the rare bacteria known to synthesize fluorinated metabolites. The genome consists of two linear replicons. The genes involved in fluorine metabolism and in the biosynthesis of the antibiotic thienamycin were mapped on both replicons.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Análise de Sequência de DNA , Streptomyces/genética , Antibacterianos/metabolismo , Cefamicinas/metabolismo , Hidrocarbonetos Fluorados/metabolismo , Dados de Sequência Molecular , Replicon , Streptomyces/isolamento & purificação , Streptomyces/metabolismo , Tienamicinas/metabolismoRESUMO
The dapdiamides make up a family of antibiotics that have been presumed to be cleaved in the target cell to enzyme-inhibitory N-acyl-2,3-diaminopropionate (DAP) warheads containing two alternative electrophilic moieties. Our prior biosynthetic studies revealed that an eneamide warhead is made first and converted to an epoxyamide via a three-enzyme branch pathway. Here we provide a rationale for this logic. We report that the R,R-epoxyamide warhead is a more efficient covalent inactivator of glucosamine-6-phosphate synthase by 1 order of magnitude versus the eneamide, and this difference correlates with a >10-fold difference in antibiotic activity for the corresponding acyl-DAP dipeptides.
Assuntos
Amidas/química , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Toxinas Bacterianas/biossíntese , Compostos de Epóxi/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Pantoea/efeitos dos fármacos , Amidas/farmacologia , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacologia , Dipeptídeos/biossíntese , Dipeptídeos/química , Compostos de Epóxi/farmacologia , Erwinia amylovora/efeitos dos fármacos , Erwinia amylovora/enzimologia , Erwinia amylovora/crescimento & desenvolvimento , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/enzimologia , Escherichia coli K12/crescimento & desenvolvimento , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/biossíntese , Pantoea/enzimologia , Pantoea/crescimento & desenvolvimento , EstereoisomerismoRESUMO
Glucosamine-6P synthase, which catalyzes glucosamine-6P synthesis from fructose-6P and glutamine, channels ammonia over 18Å between its glutaminase and synthase active sites. The crystal structures of the full-length Escherichia coli enzyme have been determined alone, in complex with the first bound substrate, fructose-6P, in the presence of fructose-6P and a glutamine analog, and in the presence of the glucosamine-6P product. These structures represent snapshots of reaction intermediates, and their comparison sheds light on the dynamics of catalysis. Upon fructose-6P binding, the C-terminal loop and the glutaminase domains get ordered, leading to the closure of the synthase site, the opening of the sugar ring and the formation of a "closed" ammonia channel. Then, glutamine binding leads to the closure of the Q-loop to protect the glutaminase site, the activation of the catalytic residues involved in glutamine hydrolysis, the swing of the side chain of Trp74, which allows the communication between the two active sites through an "open" channel, and the rotation of the glutaminase domains relative to the synthase domains dimer. Therefore, binding of the substrates at the appropriate reaction time is responsible for the formation and opening of the ammonia channel and for the activation of the enzyme glutaminase function.
Assuntos
Escherichia coli/enzimologia , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação ProteicaRESUMO
Glucosamine-6-phosphate synthase (GlmS) is responsible for the first and rate-limiting step in the hexosamine biosynthetic pathway. It catalyzes the conversion of D-fructose-6P (F6P) into D-glucosamine-6P (GlcN6P) using L-glutamine (Gln) as nitrogen donor (synthase activity) according to an ordered bi-bi process where F6P binds first. In the absence of F6P, the enzyme exhibits a weak hydrolyzing activity of Gln into Glu and ammonia (glutaminase activity), whereas the presence of F6P strongly stimulates it (hemi-synthase activity). Until now, these different activities were indirectly measured using either coupled enzyme or colorimetric methods. In this work, we have developed a direct assay monitoring the heat released by the reaction. Isothermal titration calorimetry and differential scanning calorimetry were used to determine kinetic and thermodynamic parameters of GlmS. The direct determination at 37 degrees C of kinetic parameters and affinity constants for both F6P and Gln demonstrated that part of the ammonia produced by Gln hydrolysis in the presence of both substrates is not used for the formation of the GlcN6P. The full characterization of this phenomenon allowed to identify experimental conditions where this leak of ammonia is negligible. Enthalpy measurements at 25 degrees C in buffers of various heats of protonation demonstrated that no proton exchange with the medium occurred during the enzyme-catalyzed glutaminase or synthase reaction suggesting for the first time that both products are released as a globally neutral pair composed by the Glu carboxylic side chain and the GlcN6P amine function. Finally we showed that the oligomerization state of GlmS is concentration-dependent.
Assuntos
Escherichia coli/enzimologia , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Varredura Diferencial de Calorimetria , Calorimetria Indireta , Catálise , Proteínas de Escherichia coli , Glucosamina/análogos & derivados , Glucosamina/química , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/química , Temperatura Alta , CinéticaRESUMO
The parallel synthesis of O-aryloxyamines remains an unfulfilled need in the field of medicinal chemistry and fragment-based approaches. To fill this gap a solution-phase two-step process based on (1) a copper-catalyzed cross-coupling of aryl boronic acids with a fluorous tagged N-hydroxyphthalimide, and (2) a supported aminolysis was designed and optimized using Taguchi's method. A library of O-aryloxyamines was synthesized in high yields with high purity and diversity.
Assuntos
Aminas/síntese química , Técnicas de Química Combinatória , Hidrocarbonetos Fluorados/química , Ftalimidas/química , Aminas/química , Ácidos Borônicos/química , Catálise , Cobre/química , Estrutura Molecular , EstereoisomerismoRESUMO
L-Glutamine:d-fructose-6-phosphate amidotransferase, also known as glucosamine-6-phosphate synthase (GlcN6P synthase), which catalyzes the first step in a pathway leading to the formation of uridine 5'-diphospho-N-acetyl-d-glucosamine (UDP-GlcNAc), is a key point in the metabolic control of the biosynthesis of amino sugar-containing macromolecules. The molecular mechanism of the reaction catalyzed by GlcN6P synthase is complex and involves amide bond cleavage followed by ammonia channeling and sugar isomerization. This article provides a comprehensive overview of the present knowledge on this multi-faceted enzyme emphasizing the progress made during the last five years.
Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Modelos Moleculares , Uridina Difosfato N-Acetilglicosamina/metabolismo , Amônia/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Candida albicans/enzimologia , Catálise , Ativação Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/biossíntese , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Humanos , Isomerismo , Cinética , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The enzyme glucosamine-6-phosphate synthase (GlmS) is an important point of metabolic control in biosynthesis of amino sugar-containing macromolecules and is therefore a potential target in order to design antibacterial and antifungal drugs. It has two oligomerization states, which are the active dimer and the inactive hexamer. For the first time, the potential of CE to separate and quantify the two forms was studied. After incubating GlmS with the d-glucosamine 6-phosphate (GlcN6P) inhibitor, an electrolyte based on sodium phosphate at pH 7.2 and an ionic strength of 100mM plus GlcN6P (either 2 or 20mM) allowed the hexamer-dimer separation. However, the displacement of the dimer/hexamer equilibrium during the analysis time prevented any improvement of the resolution when varying the effective separation length or the temperature of the analysis. Therefore, the use of a short-end CE method allowed the decrease in the analysis time to about 1min. Some parameters such as the temperature and the time of incubation and the ratio of the inhibitor and enzyme concentrations were studied. Then, it was also possible to test, very rapidly and with a very small amount, some molecules having an inhibition potential for the GlmS enzyme (arabinose-5-phosphate oxime, 2-amino-2-deoxy-d-glucitol 6-phosphate, and glucose-6-phosphate).
Assuntos
Eletroforese Capilar/métodos , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/análise , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidoresRESUMO
Ammonia transfer from the glutamine site to the fructose-6P site of bacterial glucosamine-6-phosphate synthase was studied by molecular dynamics simulations. The studies suggest a key role for Trp74, in the sealing of the hydrophobic channel connecting the two binding sites, as well as for the two Ala602 and Val605 residues, which form a narrow passage whose opening/closing constitutes an essential event in ammonia transfer. Kinetic analyses of the corresponding protein mutants confirmed our predictions. The efficiency of ammonia transfer which was close to zero in the W74A mutant was partially restored by increasing the size of the corresponding side-chain; the simulations performed on the W74A mutant suggested the formation of a hole in the channel. In the case of A602L and V605L mutants, the efficiency of ammonia transfer decreased to approximately 50% of the value of the native protein. None of the mutants were, however, able to use exogenous ammonia as a substrate.
Assuntos
Amônia/metabolismo , Simulação por Computador , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Amônia/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Escherichia coli/genética , Glucosamina/análogos & derivados , Glucosamina/química , Glucosamina/metabolismo , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Glutamina/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Hidrólise , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/metabolismo , Mutação PuntualRESUMO
Original inhibitors of HIV-1 protease based on a chiral bicyclic guanidinium scaffold linked to short peptidic mimics of the terminal protease sequences and to a lipophilic group were designed. These inhibitors prevent dimerization of the native protease by an interfacial structure at the highly conserved antiparallel beta-strand involving both the N and C termini that substantially account for dimerization. The preorganized guanidinium spacer introduces additional electrostatic hydrogen-bonding interactions with the C-terminal Phe-99 carboxylate. Lipophilic residues linked to side chains and the guanidinium scaffold are essential for dimerization inhibition as ascertained by Zhang kinetics (4, K(id) = 290 nM; 6 or 6', K(id) = 150 nM; 8, K(id) = 400 nM) combined with a circular dichroism study on the enzyme thermal stability. Remarkably, less hydrophobic compounds result in mixed dimerization (1a and 3) or active site inhibitors (5). Removal of the guanidinium hydrophobic groups leads to less active or inactive ligands.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Guanidinas/química , Inibidores da Protease de HIV/química , Protease de HIV/metabolismo , Sítios de Ligação , Dicroísmo Circular , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Estrutura Secundária de Proteína , Serina/química , Estereoisomerismo , Tirosina/químicaRESUMO
The enzyme glucosamine-6P Synthase (Gfat, L-glutamine:D-fructose-6P amidotransferase) is involved in the hexosamine biosynthetic pathway and catalyzes the formation of glucosamine-6P from the substrates d-fructose-6-phosphate and l-glutamine. In eukaryotic cells, Gfat is inhibited by UDPGlcNAc, the end product of the biochemical pathway. In this work we present the dissection of the binding and inhibition properties of this feedback inhibitor and of its fragments by a combination of STD-NMR experiments and inhibition measurements on the wild type human enzyme (hGfat) as well as on site-directed mutants. We demonstrate that the UDPGlcNAc binding site is located in the isomerase domain of hGfat. Two amino acid residues (G445 and G461) located at the bottom of the binding site are identified to play a key role in the specificity of UDPGlcNAc inhibition of hGfat activity vs its bacterial Escherichia coli counterpart. We also show that UDPGlcNAc subcomponents have distinct features: the nucleotidic moiety is entirely responsible for binding whereas the N-acetyl group is mandatory for inhibition but not for binding, and the sugar moiety acts as a linker between the nucleotidic and N-acetyl groups. Combining these structural recognition determinants therefore appears as a promising strategy to selectively inhibit hGfat, which may for example help reduce complications in diabetes.
Assuntos
Frutosefosfatos/metabolismo , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Glutamina/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Domínio Catalítico , Escherichia coli/enzimologia , Escherichia coli/genética , Retroalimentação Fisiológica , Frutosefosfatos/química , Expressão Gênica , Glucosamina/química , Glucosamina/metabolismo , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Glutamina/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Uridina Difosfato N-Acetilglicosamina/químicaAssuntos
Enzimas/análise , Sondas Moleculares/química , Ressonância Magnética Nuclear Biomolecular/métodos , Compostos Organometálicos/química , Itérbio/química , Catálise , Ativação Enzimática , Sondas Moleculares/síntese química , Compostos Organometálicos/síntese química , beta-Galactosidase/análiseRESUMO
Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using (14)N versus (15)N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.
Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Substituição de Aminoácidos , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Glutamina/química , Glutamina/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Isótopos de Nitrogênio , Multimerização ProteicaRESUMO
AIMS: We evaluated biological activity in leukemia cells lines of R and S enantiomers of tert-butyl 4-[(3-nitrophenoxy)-methyl]-2,2-dimethyloxazolidine-3-carboxylate (BNDC). MAIN METHODS: Cytotoxic activity was assessed by MTT assay. Flow cytometry assays were used to determined DNA fragmentation (Propidium Iodide-PI staining) and phosphatidylserine exposure (Annexin-V and PI staining). DNA condensation was evaluated by fluorescence microscopy using double-staining in leukemia cells (Hoechst and PI). Caspase activities were measured using Z-VAD-FMK, a non-selective caspase inhibitor, by flow cytometry and Z-DEVD-AMC, a selective caspase-3 substrate, by fluorescence spectrometry. KEY FINDINGS: Both enantiomers displayed cytotoxic activity against leukemia cell lines (HL60, HL60.Bcl-2, HL60.Bcl-XL and Jurkat) with low toxicity against human peripheral blood mononuclear cell--PBMC based on IC50 values. In HL60 cell lines, compounds induce exposure of phosphatidylserine and DNA fragmentation, which could be blocked by pretreatment of cells with Z-VAD-FMK. Confirming this observation, both enantiomers induced caspase-3 activation. Additional analysis revealed an increased percentage of apoptotic cells (defined as those with fragmented nuclei and condensed chromatin) after treatment with compounds. SIGNIFICANCE: Taken together, the results indicate that BNDC compounds exhibited cytotoxic and pro-apoptotic activities and have a potential for developing a new class of anticancer drugs.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Leucemia/tratamento farmacológico , Oxazóis/síntese química , Oxazóis/farmacologia , Benzimidazóis , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Descoberta de Drogas , Citometria de Fluxo , Corantes Fluorescentes , Células HL-60 , Humanos , Indicadores e Reagentes , Células Jurkat , Leucemia/patologia , Microscopia de Fluorescência , Fosfatidilserinas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , EstereoisomerismoRESUMO
The large protein motions of the bacterial enzyme glucosamine-6-phosphate synthase have been addressed using full atom normal modes analysis for the empty, the glucose-6-phosphate and the glucose-6-phosphate+glutamate bound proteins. The approach that was used involving energy minimizations along the normal modes coordinates identified functional motions of the protein, some of which were characterized earlier by X-ray diffraction studies. This method made it possible for the first time to highlight significant energy differences according to whether none, only one or both of the active sites of the protein were occupied. Our data favoured a specific motion of the glutamine binding domain following the fixation of fructose-6-phosphate and suggested a rigidified structure with both sites occupied. Here, we show that most of the collective large amplitude motions of glucosamine-6-phosphate synthase that are modulated by ligand binding are crucial for the enzyme catalytic cycle, as they strongly modify the geometry of both the ammonia channel and the C-tail, demonstrating their role in ammonia transfer and ligand binding.
Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Movimento (Física) , Amônia/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Frutosefosfatos/metabolismo , Ligantes , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Nanosized contrast agents have great potential in magnetic resonance molecular imaging applications for clinical diagnosis. This study proposes new nanoparticles spontaneously formed under mild conditions and composed of a noncovalent adduct between a gadolinium complex, a polymer of beta-cyclodextrin (pbetaCD: MW 1.5 x 10(6) g mol(-1)) and a dextran grafted with alkyl chains (MD). The formation of this supramolecular nanoassembly is based upon a "lock-and-key" recognition process in which the hydrophobic alkyl chains of MD and the adamantyl moieties of macrocyclic Gd(III) chelates are included in the cavities of pbetaCD. The large number of betaCDs contained in the pbetaCD resulted in the formation of 200 nm diameter nanoparticles, each entrapping 1.8 x 10(5) molecules of a low-molecular-weight Gd complex. This system, which exhibits a great relaxivity enhancement (48.4 mM(-1) s(-1), at 20 MHz and 37 degrees C) compared to the Gd(III) chelate itself (5.2 mM(-1) s(-1)), appears to be a promising strategy for the in vivo targeted delivery of Gd(III) complexes. The mechanisms of particle formation, conjugation strategies, and relaxometric characterizations in the field of contrast-enhanced magnetic resonance imaging are discussed.
Assuntos
Quelantes/química , Meios de Contraste/química , Dextranos/química , Gadolínio/química , Imageamento por Ressonância Magnética , beta-Ciclodextrinas/química , Quelantes/síntese química , Substâncias Macromoleculares/química , Estrutura Molecular , Nanopartículas/química , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Polímeros/químicaRESUMO
Glutamine:fructose-6-phosphate amidotransferase (Gfat) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway. The increasing amount of evidence that links excess hexosamine biosynthesis with pathogenic complications of type II diabetes highlights the need to understand the regulation of Gfat. Previous studies showed that eukaryotic Gfat is subjected to feedback inhibition by UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and to phosphorylation by cAMP-activated protein kinase A (PKA). In this study, overexpression of human Gfat isoform 1 (hGfat1) in insect cells revealed that hGfat1 is phosphorylated in vivo. Using matrix-assisted laser desorption/ionization and electrospray tandem mass spectrometry, we have identified Ser243 as a novel phosphorylation site. Biochemical properties of the wild type and the Ser243Glu mutant of hGfat1 overexpressed in Escherichia coli were compared. Our results provide evidence that phosphorylation at Ser243 stimulates glucosamine 6-phosphate-synthesizing activity, lowers amidohydrolyzing activity in the absence of fructose 6-phosphate (F6P) (glutaminase activity), and lowers Km(F6P) 2-fold, but has no effect on UDP-GlcNAc inhibition. On the basis of the sequence consensus, AMP-activated protein kinase and calcium/calmodulin-dependent kinase II were identified to phosphorylate specifically Ser243 in vitro. Phosphorylation by these two kinases results in an increase of enzymatic activity by 1.4-fold. These findings suggest for the first time that hGfat1 may be regulated by kinases other than PKA.