RESUMO
Glioblastoma multiforme is the most aggressive type of glioma, with limited treatment and poor prognosis. Despite some advances over the last decade, validation of novel and selective antiglioma agents remains a challenge in clinical pharmacology. Prior studies have shown that leguminous lectins may exert various biological effects, including antitumor properties. Accordingly, this study aimed to evaluate the mechanisms underlying the antiglioma activity of ConBr, a lectin extracted from the Canavalia brasiliensis seeds. ConBr at lower concentrations inhibited C6 glioma cell migration while higher levels promoted cell death dependent upon carbohydrate recognition domain (CRD) structure. ConBr increased p38MAPK and JNK and decreased ERK1/2 and Akt phosphorylation. Moreover, ConBr inhibited mTORC1 phosphorylation associated with accumulation of autophagic markers, such as acidic vacuoles and LC3 cleavage. Inhibition of early steps of autophagy with 3-methyl-adenine (3-MA) partially protected whereas the later autophagy inhibitor Chloroquine (CQ) had no protective effect upon ConBr cytotoxicity. ConBr also augmented caspase-3 activation without affecting mitochondrial function. Noteworthy, the caspase-8 inhibitor IETF-fmk attenuated ConBr induced autophagy and C6 glioma cell death. Finally, ConBr did not show cytotoxicity against primary astrocytes, suggesting a selective antiglioma activity. In summary, our results indicate that ConBr requires functional CRD lectin domain to exert antiglioma activity, and its cytotoxicity is associated with MAPKs and Akt pathways modulation and autophagy- and caspase-8- dependent cell death.
Assuntos
Antineoplásicos/farmacologia , Caspase 8/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glioma/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glioma/metabolismo , Glioma/patologia , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Polissacarídeos/metabolismo , Domínios Proteicos/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RatosRESUMO
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones which prevent the nonspecific aggregation of non-native proteins. Five potential sHsps exist in the parasite Toxoplasma gondii. They are located in different intracellular compartments including mitochondria and are differentially expressed during the parasite's life cycle. Here, we analyzed the structural and functional properties of all five proteins. Interestingly, this first in vitro characterization of sHsps from protists showed that all T. gondii sHsps exhibit the characteristic properties of sHsps such as oligomeric structure and chaperone activity. However, differences in their quaternary structure and in their specific chaperone properties exist. On the structural level, the T. gondii sHsps can be divided in small (12-18 subunits) and large (24-32 subunits) oligomers. Furthermore, they differ in their interaction with non-native proteins. While some bind substrates tightly, others interact more transiently. The chaperone activity of the three more mono-disperse T. gondii sHsps is regulated by temperature with a decrease in temperature leading to the activation of chaperone activity, suggesting an adaption to specific steps of the parasite's life cycle.
Assuntos
Proteínas de Choque Térmico Pequenas/genética , Filogenia , Proteínas de Protozoários/genética , Toxoplasma/genética , Motivos de Aminoácidos/fisiologia , Animais , Proteínas de Choque Térmico Pequenas/metabolismo , Estrutura Quaternária de Proteína/fisiologia , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Toxoplasma/metabolismoRESUMO
Interleukin-22 (IL-22) plays an important role in the regulation of immune and inflammatory responses in mammals. The IL-22 binding protein (IL-22BP), a soluble receptor that specifically binds IL-22, prevents the IL-22/interleukin-22 receptor 1 (IL-22R1)/interleukin-10 receptor 2 (IL-10R2) complex assembly and blocks IL-22 biological activity. Here we present the crystal structure of the IL-22/IL-22BP complex at 2.75 A resolution. The structure reveals IL-22BP residues critical for IL-22 binding, which were confirmed by site-directed mutagenesis and functional studies. Comparison of IL-22/IL-22BP and IL-22/IL-22R1 crystal structures shows that both receptors display an overlapping IL-22 binding surface, which is consistent with the inhibitory role played by IL-22 binding protein.
Assuntos
Interleucinas/química , Receptores de Interleucina/química , Sítios de Ligação/fisiologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Subunidade beta de Receptor de Interleucina-10/química , Subunidade beta de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Interleucina 22RESUMO
The common observation that cancer cells present higher glycolytic rates when compared to control cells leads to the proposal of glycolysis as a potential target for the development of anti-tumoral agents. Anti-inflammatory drugs, such as acetylsalicylic acid (ASA) and salicylic acid (SA), present anti-tumoral properties, inducing apoptosis and altering tumor glucose utilization. The present work aims at evaluating whether ASA could directly decrease cell glycolysis through inhibition of the major regulatory enzyme within this pathway, 6-phosphofructo-1-kinase (PFK). We show that ASA and SA inhibit purified PFK in a dose-dependent manner, and that this inhibition occurs due to the modulation of the enzyme quaternary structure. ASA and SA promote the dissociation of the enzyme active tetramers into quite inactive dimers, a common regulatory mechanism of this enzyme. The inhibitory effects of ASA and SA on PFK are fully reversible and can be prevented or reverted by the binding of the enzyme to the actin filaments. Both drugs are also able to decrease glucose consumption by human breast cancer cell line MCF-7, as well as its viability, which decrease parallelly to the inhibition of PFK on these cells. In the end, we demonstrate the ability of ASA and SA to directly modulate an important regulatory intracellular enzyme, and propose that this is one of their mechanisms promoting anti-tumoral effects.
Assuntos
Aspirina/farmacologia , Glucose/antagonistas & inibidores , Fosfofrutoquinase-1/antagonistas & inibidores , Fosfofrutoquinase-1/química , Estrutura Quaternária de Proteína/efeitos dos fármacos , Ácido Salicílico/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Glucose/metabolismo , Humanos , Fosfofrutoquinase-1/metabolismo , Estrutura Quaternária de Proteína/fisiologia , Coelhos , Relação Estrutura-AtividadeRESUMO
Xylans are the most abundant polysaccharides forming the plant cell wall hemicelluloses, and they are degraded, among other proteins, by beta-xylosidase enzymes. In this work, the structural and biophysical properties of the family 52 beta-xylosidase from Geobacillus stearothermophilus, XynB2, are described. Size exclusion chromatography, analytical centrifugation, ITC, CD, fluorescence (steady state and ANS-binding) and FTIR were used to obtain the structure, the oligomerization state and the conformational changes of XynB2, as pH, chemical denaturants or temperature were modified. This report describes the first extensive conformational characterization of a family 52 beta-xylosidase. The active protein was a highly hydrated dimer, whose active site was formed by the two protomers, and it probably involved aromatic residues. At low pH, the protein was not active and it populated a monomeric molten-globule-like species, which had a conformational transition with a pK(a) of approximately 4.0. Thermal and chemical-denaturations of the native protein showed hysteresis behaviour. The protein at physiological pH was formed by alpha-helix (30%) and beta-sheet (30%), as shown by CD and FTIR. Comparison with other xylosidases of the same family indicates that the percentages of secondary structure seem to be conserved among the members of the family.
Assuntos
Bacillaceae/enzimologia , Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Dicroísmo Circular/métodos , Dimerização , Concentração de Íons de Hidrogênio , Estrutura Quaternária de Proteína/fisiologia , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Methodologies and results of studies on the kinetics of peroxiredoxins (Prx) are reviewed. Peroxiredoxins are broad-spectrum peroxidases that catalyze the reduction of H2O2, organic hydroperoxides and peroxynitrite by thiols. Their catalytic cycle starts with the oxidation of a particularly reactive cysteine residue (C(P)) to a sulfenic acid derivative by the peroxide substrate, the sulfenic acid then reacts with a thiol to form a disulfide, and the cycle is completed by thiol/disulfide exchange reactions that regenerate the ground-state enzyme. Depending on the subtype of peroxiredoxin, the thiol reacting with the primary oxidation product (E-SOH) may be a cysteine residue of a second subunit (typical 2-Cys Prx), a cysteine residue of the same subunit (atypical 2-Cys Prx) or reducing substrate (1-Cys Prx and at least one example of an atypical 2-Cys Prx). In a typical 2-Cys Prx the intra-subunit disulfide formation with the second "resolving" cysteine (C(R)) is mandatory for the reduction by the specific substrate, which is a protein characterized by a CXXC motif such as thioredoxin, tryparedoxin or AhpF. These consecutive redox reactions define the catalysis as an enzyme substitution mechanism, which is corroborated by a ping-pong pattern that is commonly observed in steady-state analyses, chemical identification of catalytic intermediates and stopped-flow analyses of partial reactions. More complex kinetic patterns are discussed in terms of cooperativity between the subunits of the oligomeric enzymes, generation of different oxidized intermediates or partial over-oxidation of C(P) to a sulfinic acid. Saturation kinetics is often not observed indicating that a typical complex between reduced enzyme and hydroperoxide is not formed and that, in these cases, formation of the complex between the oxidized enzyme and its reducing substrate is slower than the reaction within this complex. Working with sulphur catalysis, Prxs are usually less efficient than the heme- or selenium-containing peroxidases, but in some cases the k(+1) values (bimolecular rate constant for oxidation of reduced E by ROOH) are comparable, the overall range being 2 x 10(3)-4 x 10(7) M(-1)s(-1) depending on the hydroperoxide and the individual Prx. For the reduction of peroxynitrite k(+1) values of 1 x 10(6) up to 7 x 10(7) M(-1)s(-1) have been measured. The net forward rate constants k'(+2) for the reductive part of the cycle range between 2 x 10(4)-1 x 10(7) M(-1)s(-1). These kinetic characteristics qualify the peroxiredoxins as moderately efficient devices to detoxify hydroperoxides, which is pivotal to organisms devoid of more efficient peroxidases, and as most relevant to the detoxification of peroxynitrite. In higher organisms, their specific role is seen in the regulation of signalling cascades that are modulated by H2O2, lipid hydroperoxides or peroxynitrite.
Assuntos
Peróxidos/química , Peróxidos/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Catálise , Domínio Catalítico/fisiologia , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Cinética , Metaloproteínas/química , Metaloproteínas/metabolismo , Oxirredução , Estrutura Quaternária de Proteína/fisiologia , Especificidade por Substrato/fisiologia , Ácidos Sulfênicos/química , Ácidos Sulfênicos/metabolismoRESUMO
The alphaM4 transmembrane domain of the nicotinic acetylcholine receptor (AChR) is flanked by two basic amino acids (His(408) and Arg(429)) located at its cytoplasmic- and extracellular-facing extremes, respectively, at the level of the phospholipid polar head regions of the postsynaptic membrane. A series of single and double alphaM4 mutants (His(408)Ala, Arg(429)Ala, Arg(429)Glu, His(408)Ala/Arg(429)Ala, and His(408)Ala/Arg(429)Glu) of the adult muscle-type AChR were produced and coexpressed with wild-type beta, delta, and epsilon subunits as stable clones in a mammalian heterologous expression system (CHO-K1 cells). The mutants were studied by alpha-bungarotoxin ([(125)I]alpha-BTX) binding, fluorescence microscopy, and equilibrium sucrose gradient centrifugation. Cell-surface [(125)I]alpha-BTX binding diminished approximately 40% in His(408)Ala and as much as 95% in the Arg(429)Ala mutant. Reversing the amino acid charge (e.g., Arg(429)Glu) abolished cell-surface expression of AChR. Fluorescence microscopy disclosed that AChR was retained at the endoplasmic reticulum, with an enhanced occurrence of unassembled AChR species in the mutant clones. Centrifugation analysis confirmed the lack of fully assembled AChR pentamers in all mutants with the exception of His(408)Ala. We conclude that His(408) and Arg(429) in alphaM4 are involved in assembly and cell-surface targeting of muscle AChR. Arg(429) plays a more decisive role in these two processes, suggesting an asymmetric weight of the charged motifs at each extreme of the alpha subunit M4 transmembrane segment. (c) 2006 Wiley-Liss, Inc.
Assuntos
Motivos de Aminoácidos/fisiologia , Músculo Esquelético/química , Estrutura Quaternária de Proteína/fisiologia , Receptores Nicotínicos/química , Sequência de Aminoácidos , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Sequência Conservada , Cricetinae , Cricetulus , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Músculo Esquelético/fisiologia , Ligação Proteica , Receptores Nicotínicos/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Thyroid hormone nuclear receptors (TRs) bind to DNA and activate transcription as heterodimers with the retinoid X receptor (RXR) or as homodimers or monomers. RXR also binds to DNA and activates transcription as homodimers but can, in addition, self-associate into homotetramers in the absence of ligand and DNA templates. It is thought that homotetramer formation serves to sequester excess RXRs into an inactive pool within the cell. Here, we report systematic studies of the multimeric state of a recombinant human TRbeta1 truncation (hTRbeta1deltaAB) that encompasses the complete DNA binding domain and ligand binding domain in solution. Native gel electrophoresis, chemical crosslinking, gel filtration, and dynamic light scattering experiments reveal that hTRbeta1deltaAB forms a mixture of monomers, dimers, and tetramers. Like RXR, increasing protein concentration shifts the equilibrium between TR multimers toward tetramer formation, whereas binding of cognate thyroid hormone leads to dissociation of tetramers and increased formation of dimers. This work represents the first evidence that apo-hTRbeta1 forms homotetramers. The findings raise the possibility that tetramer formation provides an additional, and previously unsuspected, level of control of TR activity and that the capacity for homotetramer formation may be more widespread in the nuclear receptor family than previously thought.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Receptores beta dos Hormônios Tireóideos/química , Receptores beta dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/química , Tri-Iodotironina/metabolismo , Sequência de Aminoácidos/genética , DNA/química , DNA/fisiologia , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ligantes , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/fisiologia , Proteínas Recombinantes/genética , Receptores X de Retinoides , Soluções/química , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Folding of glycoproteins entering the secretory pathway is strictly surveyed in the endoplasmic reticulum by a quality control system. Folding intermediates and proteins irreparably misfolded are marked via glucosylation by the UDPglucose:glycoprotein glucosyltransferase, an enzyme that acts as a folding sensor by exclusively labeling glycoproteins not displaying their native structures. Here we show that this sensing mechanism also applies to the oligomerization of protein complexes, as the glucosyltransferase appeared to be able to glucosylate folded complex subunits lacking the full complement of oligomer components.