RESUMO
The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [3H]-carnitineex/carnitinein antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC50 derived from the dose-response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.
Assuntos
Patulina , Humanos , Animais , Ratos , Escherichia coli/metabolismo , Cisteína/metabolismo , Reagentes de Sulfidrila/farmacologia , Carnitina/farmacologia , Carnitina/metabolismo , Glutationa/metabolismo , Proteínas de Membrana TransportadorasRESUMO
Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most important parts of the mitochondrial permeability transition pore are F1F0 ATPase, the adenine nucleotide translocase (ANT), and the inorganic phosphate cotransporter. The ANT conformation play a significant role in the Tl+-induced MPTP opening in the inner membrane of calcium-loaded rat liver mitochondria. The present study tests the effects of a lysine reagent, pyridoxal 5-phosphate (PLP), and thiol reagents (phenylarsine oxide, tert-butylhydroperoxide, eosin-5-maleimide, and mersalyl) to induce the MPTP opening that was accompanied by increased swelling, membrane potential decline, and decreased respiration in 3 and 3UDNP (2,4-dinitrophenol uncoupled) states. This pore opening was more noticeable in increasing the concentration of PLP and thiol reagents. However, more significant concentrations of PLP were required to induce the above effects comparable to those of these thiol reagents. This study suggests that the Tl+-induced MPTP opening can be associated not only with the state of functionally active cysteines of the pore parts, but may be due to a change in the state of the corresponding lysines forming the pore structure.
Assuntos
Lisina , Poro de Transição de Permeabilidade Mitocondrial , Animais , Ratos , Cálcio/metabolismo , Indicadores e Reagentes , Lisina/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Permeabilidade , Fosfato de Piridoxal/farmacologia , Fosfato de Piridoxal/metabolismo , Ratos Wistar , Reagentes de Sulfidrila/farmacologia , Tálio/farmacologiaRESUMO
BACKGROUND: Gram-negative and Gram-positive bacteria produce beta-lactamase as factors to overcome beta-lactam antibiotics, causing their hydrolysis and impaired antimicrobial action. Class A beta-lactamase contains the chromosomal sulfhydryl reagent variable (SHV, point mutation variants of SHV-1), LEN (Klebsiella pneumoniae strain LEN-1), and other K. pneumoniae beta-lactamase (OKP) found mostly in Klebsiella's phylogroups. The SHV known as extended-spectrum ß-lactamase can inactivate most beta-lactam antibiotics. Class A also includes the worrisome plasmid-encoded Klebsiella pneumoniae carbapenemase (KPC-2), a carbapenemase that can inactivate most beta-lactam antibiotics, carbapenems, and some beta-lactamase inhibitors. OBJECTIVES: So far, there is no 3D crystal structure for OKP-B, so our goal was to perform structural characterisation and molecular docking studies of this new enzyme. METHODS: We applied a homology modelling method to build the OKP-B-6 structure, which was compared with SHV-1 and KPC-2 according to their electrostatic potentials at the active site. Using the DockThor-VS, we performed molecular docking of the SHV-1 inhibitors commercially available as sulbactam, tazobactam, and avibactam against the constructed model of OKP-B-6. FINDINGS: From the point of view of enzyme inhibition, our results indicate that OKP-B-6 should be an extended-spectrum beta-lactamase (ESBL) susceptible to the same drugs as SHV-1. MAIN CONCLUSIONS: This conclusion advantageously impacts the clinical control of the bacterial pathogens encoding OKP-B in their genome by using any effective, broad-spectrum, and multitarget inhibitor against SHV-containing bacteria.
Assuntos
Sulbactam , Inibidores de beta-Lactamases , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Klebsiella , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Sulbactam/farmacologia , Reagentes de Sulfidrila/farmacologia , Tazobactam/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
Thiol reagents dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) are sulfhydryl reagents that can be used to disperse cold autoagglutinins coating red blood cells (RBCs). DTT and 2-ME are primarily used when warm washing of the coated RBCs fails to successfully disperse the cold autoantibody. Using a weak concentration of DTT or 2-ME, the cold IgM agglutinin can be removed from the coated RBCs without disrupting the IgG or complement coating the RBCs. The treated RBCs can be used for ABO typing, antigen typing, or the direct antiglobulin test.
Assuntos
Aglutinação , Eritrócitos , Teste de Coombs , Ditiotreitol , Humanos , Mercaptoetanol , Reagentes de SulfidrilaRESUMO
Recent data have shown that the mitochondrial permeability transition pore (MPTP) is the complex of the Ca2+-modified adenine nucleotide translocase (ANT) and the Ca2+-modified ATP synthase. We found in a previous study that ANT conformational changes may be involved in Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat liver mitochondria. In this study, the effects of thiol-modifying agents (eosin-5-maleimide (EMA), fluorescein isothiocyanate (FITC), Cu(o-phenanthroline)2 (Cu(OP)2), and embelin (Emb)), and MPTP inhibitors (ADP, cyclosporine A (CsA), n-ethylmaleimide (NEM), and trifluoperazine (TFP)) on MPTP opening were tested simultaneously with increases in swelling, membrane potential (ΔΨmito) decline, decreases in state 3, 4, and 3UDNP (2,4-dinitrophenol-uncoupled) respiration, and changes in the inner membrane free thiol group content. The effects of these thiol-modifying agents on the studied mitochondrial characteristics were multidirectional and showed a clear dependence on their concentration. This research suggests that Tl+-induced MPTP opening in the inner membrane of calcium-loaded mitochondria may be caused by the interaction of used reagents (EMA, FITC, Emb, Cu(OP)2) with active groups of ANT, the mitochondrial phosphate carrier (PiC) and the mitochondrial respiratory chain complexes. This study provides further insight into the causes of thallium toxicity and may be useful in the development of new treatments for thallium poisoning.
Assuntos
Mitocôndrias Hepáticas , Proteínas de Transporte da Membrana Mitocondrial , Reagentes de Sulfidrila , Tálio , Animais , Cálcio/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Fígado , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/farmacologia , Tálio/farmacologiaRESUMO
The effects of both Tl+ and thiol reagents were studied on the content of the inner membrane free SH-groups, detected with Ellman reagent, and the inner membrane potential as well as swelling and respiration of succinate-energized rat liver mitochondria in medium containing TlNO3 and KNO3. These effects resulted in a rise in swelling and a decrease in the content, the potential, and mitochondrial respiration in 3 and 2,4-dinitrophenol-uncoupled states. A maximal effect was seen when phenylarsine oxide reacting with thiol groups recessed into the hydrophobic regions of the membrane. Compared with phenylarsine oxide, the effective concentrations of other reagents were approximately one order of magnitude higher in experiments with mersalyl and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and two orders of magnitude higher in experiments with tert-butyl hydroperoxide and diamide. The above effects of Tl+ and the thiol reagents became even more pronounced with calcium overload of mitochondria. However, the effects were suppressed by inhibitors of the mitochondrial permeability transition pore (cyclosporine A, ADP, and n-ethylmaleimide). These findings suggest that opening of the pore induced by Tl+ in the inner membrane can be dependent on the conformation state of the adenine nucleotide translocase, which depends on the activity of its thiol groups.
Assuntos
Mitocôndrias Hepáticas , Proteínas de Transporte da Membrana Mitocondrial , Animais , Cálcio/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/farmacologia , Permeabilidade , Ratos , Ratos Wistar , Respiração , Ácido Succínico/metabolismo , Ácido Succínico/farmacologia , Compostos de Sulfidrila/metabolismo , Compostos de Sulfidrila/farmacologia , Reagentes de Sulfidrila/metabolismo , Reagentes de Sulfidrila/farmacologia , Tálio/metabolismo , Tálio/farmacologiaRESUMO
Cultivar-dependent cadmium (Cd) accumulation was principal in developing Cd-pollution safe cultivars (PSCs). Proteins related to different Cd accumulations of the low-Cd-accumulating (SJ19) and high-Cd-accumulating (CX4) cultivars were investigated by iTRAQ analysis. Higher Cd bioaccumulation factors and translocation factor in CX4 than in SJ19 were consistent with the cultivar-dependent Cd accumulations. The Cd uptake was promoted in CX4 due to its higher expression of Cd-binding proteins and the lower expression of Cd-efflux proteins in roots. What's more, significantly elevated thiol groups (PC2 and PC3) in CX4 under Cd stress might contribute to the high Cd accumulation in roots and the root-to-shoot translocation of Cd-PC complex. Up-regulated proteins involved in cellulose biosynthesis and pectin de-esterification in SJ19 enhanced the Cd sequestration of root cell walls, which was considered as the predominant strategy for reducing Cd accumulation in shoots. The present study provided novel insights in the cultivar-dependent Cd accumulation in shoots of B. parachinensis.
Assuntos
Brassica/metabolismo , Cádmio/metabolismo , Proteínas de Plantas/metabolismo , Poluentes do Solo/metabolismo , Transporte Biológico , Brassica/genética , Celulose/metabolismo , Pectinas/metabolismo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Proteômica , Reagentes de Sulfidrila/metabolismoRESUMO
The transcription factor Spx is the master regulator of the disulfide stress response in Bacillus subtilis. Intriguingly, the activation of Spx by diamide relies entirely on posttranslational regulatory events in spite of the complex transcriptional control of the spx gene. Here, we show that cell wall stress, but not membrane stress, also results in induction of the Spx regulon. Remarkably, two major differences were found regarding the mechanism of induction of Spx under cell wall stress in comparison to disulfide stress. First, transcriptional induction of the spx gene from a σM -dependent promoter is required for accumulation of Spx in response to cell wall stress. Second, activation of the Spx regulon during cell wall stress is not accompanied by oxidation of the Spx disulfide switch. Finally, we demonstrate that cells lacking Spx have increased sensitivity toward antibiotics inhibiting both early and late steps in peptidoglycan synthesis, suggesting that the Spx regulon plays an important adaptive role in the cell wall stress response. This study expands the functional role of the Spx regulon and reveals novel regulatory mechanisms that result in induction of Spx in B. subtilis.
Assuntos
Bacillus subtilis/genética , Estresse Oxidativo , Regulon , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/fisiologia , Diamida/farmacologia , Dissulfetos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos , Estresse Oxidativo/efeitos dos fármacos , Regiões Promotoras Genéticas , Reagentes de Sulfidrila/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Small heat-shock proteins (sHSPs) are a conserved group of molecular chaperones with important roles in cellular proteostasis. Although sHSPs are characterized by their small monomeric weight, they typically assemble into large polydisperse oligomers that vary in both size and shape but are principally composed of dimeric building blocks. These assemblies can include different sHSP orthologues, creating additional complexity that may affect chaperone activity. However, the structural and functional properties of such hetero-oligomers are poorly understood. We became interested in hetero-oligomer formation between human heat-shock protein family B (small) member 1 (HSPB1) and HSPB6, which are both highly expressed in skeletal muscle. When mixed in vitro, these two sHSPs form a polydisperse oligomer array composed solely of heterodimers, suggesting preferential association that is determined at the monomer level. Previously, we have shown that the sHSP N-terminal domains (NTDs), which have a high degree of intrinsic disorder, are essential for the biased formation. Here we employed iterative deletion mapping to elucidate how the NTD of HSPB6 influences its preferential association with HSPB1 and show that this region has multiple roles in this process. First, the highly conserved motif RLFDQXFG is necessary for subunit exchange among oligomers. Second, a site â¼20 residues downstream of this motif determines the size of the resultant hetero-oligomers. Third, a region unique to HSPB6 dictates the preferential formation of heterodimers. In conclusion, the disordered NTD of HSPB6 helps regulate the size and stability of hetero-oligomeric complexes, indicating that terminal sHSP regions define the assembly properties of these proteins.
Assuntos
Proteínas de Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Modelos Moleculares , Motivos de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Reagentes de Ligações Cruzadas/farmacologia , Dimerização , Deleção de Genes , Proteínas de Choque Térmico HSP20/química , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Reagentes de Sulfidrila/farmacologiaRESUMO
Cardiovascular dysfunction and organ damage are hallmarks of sepsis and septic shock. Protein S-nitrosylation by nitric oxide has been described as an important modifier of protein function. We studied whether protein nitrosylation/denitrosylation would impact positively in hemodynamic parameters of septic rats. Polymicrobial sepsis was induced by cecal ligation and puncture. Female Wistar rats were treated with increasing doses of DTNB [5,5'-dithio-bis-(2-nitrobenzoic acid)] 30min before or 4 or 12h after sepsis induction. Twenty-four hours after surgery the following data was obtained: aorta response to phenylephrine, mean arterial pressure, vascular reactivity to phenylephrine, biochemical markers of organ damage, survival and aorta protein nitrosylation profile. Sepsis substantially decreases blood pressure and the response of aorta rings and of blood pressure to phenylephrine, as well as increased plasma levels of organ damage markers, mortality of 60% and S-nitrosylation of aorta proteins increased during sepsis. Treatment with DTNB 12h after septic shock induction reversed the loss of response of aorta rings and blood pressure to vasoconstrictors, reduced organ damage and protein nitrosylation and increased survival to 80%. Increases in protein S-nitrosylation are related to cardiovascular dysfunction and multiple organ injury during sepsis. Treatment of rats with DTNB reduced the excessive protein S-nitrosylation, including that in calcium-dependent potassium channels (BKCa), reversed the cardiovascular dysfunction, improved markers of organ dysfunction and glycemic profile and substantially reduced mortality. Since all these beneficial consequences were attained even if DTNB was administered after septic shock onset, protein (de)nitrosylation may be a suitable target for sepsis treatment.
Assuntos
Desnitrificação/efeitos dos fármacos , Ácido Ditionitrobenzoico/uso terapêutico , Choque Séptico/tratamento farmacológico , Reagentes de Sulfidrila/uso terapêutico , Animais , Pressão Arterial/efeitos dos fármacos , Desnitrificação/fisiologia , Modelos Animais de Doenças , Ácido Ditionitrobenzoico/farmacologia , Feminino , Nitrosação , Estresse Nitrosativo/efeitos dos fármacos , Ratos , Ratos Wistar , Choque Séptico/metabolismo , Choque Séptico/patologia , Choque Séptico/fisiopatologia , Reagentes de Sulfidrila/farmacologia , Resultado do TratamentoRESUMO
There is a dire need for new treatments for Alzheimer's disease (AD). Principal drugs have reached maturity, and the number of people affected by AD is growing at a rapid rate. After years of research and many clinical trials, only symptomatic treatments are available. An effective disease-modifying drug for AD needs to be discovered. The research presented in this paper aims to facilitate in the discovery of new potential targets that could help in the ongoing AD research. Aryl methanesulfonate derivatives were screened for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities. IC50 values between 0.660 and 3.397 µM against AChE and 0.885 and 2.596 µM against BuChE were obtained.
Assuntos
Inibidores da Colinesterase/farmacologia , Descoberta de Drogas , Mesilatos/farmacologia , Nootrópicos/farmacologia , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Ácido Ditionitrobenzoico/química , Electrophorus , Proteínas de Peixes/antagonistas & inibidores , Proteínas de Peixes/metabolismo , Cavalos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Concentração Osmolar , Espectrofotometria , Reagentes de Sulfidrila/químicaRESUMO
Thiol-based redox regulation is considered to support light-responsive control of various chloroplast functions. The redox cascade via ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) has been recognized as a key to transmitting reducing power; however, Arabidopsis thaliana genome sequencing has revealed that as many as five Trx subtypes encoded by a total of 10 nuclear genes are targeted to chloroplasts. Because each Trx isoform seems to have a distinct target selectivity, the electron distribution from FTR to multiple Trxs is thought to be the critical branch point for determining the consequence of chloroplast redox regulation. In the present study, we aimed to comprehensively characterize the kinetics of electron transfer from FTR to 10 Trx isoforms. We prepared the recombinant FTR protein from Arabidopsis in the heterodimeric form containing the Fe-S cluster. By reconstituting the FTR/Trx system in vitro, we showed that FTR prepared here was enzymatically active and suitable for uncovering biochemical features of chloroplast redox regulation. A series of redox state determinations using the thiol-modifying reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate, indicated that all chloroplast Trx isoforms are commonly reduced by FTR; however, significantly different efficiencies were evident. These differences were apparently correlated with the distinct midpoint redox potentials among Trxs. Even when the experiments were performed under conditions of hypothetical in vivo stoichiometry of FTR and Trxs, a similar trend in distinguishable electron transfers was observed. These data highlight an aspect of highly organized circuits in the chloroplast redox regulation network.
Assuntos
Proteínas de Arabidopsis/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Transporte de Elétrons , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Oxirredutases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Biocatálise/efeitos dos fármacos , Domínio Catalítico , Tiorredoxinas de Cloroplastos/química , Tiorredoxinas de Cloroplastos/genética , Cloroplastos/enzimologia , Transporte de Elétrons/efeitos dos fármacos , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estilbenos/farmacologia , Reagentes de Sulfidrila/farmacologia , Ácidos Sulfônicos/farmacologiaRESUMO
To determine the binding-site of a combinatorially-selected peptide possessing a fluoroprobe, a novel cysteine reactive small photo-crosslinker that can be excited by a conventional long-wavelength ultraviolet handlamp (365 nm) was synthesized via Suzuki coupling with three steps. The crosslinker is rationally designed, not only as a bioisostere of the fluoroprobe, but as a caged-fluorophore, and the photo-crosslinked target protein became fluorescent with a large Stokes-shift. By introducing the crosslinker to a designated sulfhydryl (SH) group of a combinatorially-selected peptide, the protein-binding site of the targeted peptide was deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/fluorescence imaging followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis.
Assuntos
Reagentes de Ligações Cruzadas/química , Corantes Fluorescentes/química , Peptídeos/química , Reagentes de Sulfidrila/química , Sítios de Ligação , Técnicas de Química Combinatória , Cisteína/química , Peptídeos/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Recently, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Although there are approximately 30â¯000 genes in the human genome, only 2% are targeted by currently approved small-molecule drugs. One reason that many targets remain neglected by drug discovery programs is the absence of biochemical assays enabling evaluation of the potency of inhibitors in a quantitative and high-throughput manner. To overcome this issue, we developed a biochemical assay to evaluate the potency of both reversible and irreversible inhibitors using a nonspecific thiol-labeling fluorescent probe. The assay can be applied to any targets with a cysteine residue in a pocket that can accommodate small-molecule ligands. By constructing a mathematical model, we showed that the potency of compounds can be quantitatively evaluated by performing an activity-based protein profiling assay. In addition, the validity of the theory was confirmed experimentally using epidermal growth factor receptor kinase as a model target. This approach provides an assay system for targets for which biochemical assays cannot be developed. Our approach can potentially not only expand the number of exploitable targets but also accelerate the lead optimization process by providing quantitative structure-activity relationship information.
Assuntos
Compostos de Boro/metabolismo , Descoberta de Drogas/métodos , Receptores ErbB/antagonistas & inibidores , Corantes Fluorescentes/metabolismo , Maleimidas/metabolismo , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Reagentes de Sulfidrila/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Biocatálise , Compostos de Boro/química , Domínio Catalítico , Cisteína/química , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Ligantes , Maleimidas/química , Conformação Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Relação Quantitativa Estrutura-Atividade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Spodoptera , Reagentes de Sulfidrila/químicaRESUMO
Submicrometer aggregates are frequently present at low levels in antibody-based therapeutics. Although intuition suggests that the fraction of the aggregate or the size of the aggregate present might correlate with deleterious clinical properties or formulation difficulties, it has been challenging to demonstrate which aggregate states, if any, trigger specific biological effects. One source of uncertainty about the putative linkage between aggregation and safety or efficacy lies in the likelihood that noncovalent aggregation differs in ideal buffers versus in serum and biological tissues; self-association or association with other proteins may vary widely with environment. Therefore, methods for monitoring aggregation and aggregate behavior in biologically relevant matrices could provide a tool for better predicting aggregate-dependent clinical outcomes and provide a basis for antibody engineering prior to clinical studies. Here, we generate models for soluble aggregates of THIOMABs and a bispecific antibody (bsAb) of defined size and exploit fluorescence correlation spectroscopy to monitor their diffusion properties in serum and viscosity-matched buffers. The monomers, dimers, and trimers of both THIOMABs and a bsAb reveal a modest increase in diffusion time in serum greater than expected for an increase in viscosity alone. A mixture of larger aggregates containing mostly bsAb pentamers exhibits a marked increase in diffusion time in serum and much greater intrasample variability, consistent with significant aggregation or interactions with serum components. The results indicate that small aggregates of several IgG platforms are not likely to aggregate with serum components, but nanometer-scale aggregates larger than trimers can interact with the serum in an Ab-dependent manner.
Assuntos
Anticorpos Biespecíficos/química , Proteínas Sanguíneas/química , Imunoglobulina G/química , Agregados Proteicos , Trastuzumab/química , Algoritmos , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/análise , Anticorpos Biespecíficos/genética , Proteínas Sanguíneas/análise , Reagentes de Ligações Cruzadas/farmacologia , Difusão , Ditiotreitol/farmacologia , Composição de Medicamentos , Glutaral/farmacologia , Humanos , Hidrodinâmica , Imunoglobulina G/efeitos adversos , Imunoglobulina G/análise , Imunoglobulina G/genética , Peso Molecular , Tamanho da Partícula , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Reprodutibilidade dos Testes , Solubilidade , Reagentes de Sulfidrila/farmacologia , Trastuzumab/efeitos adversos , Trastuzumab/análise , ViscosidadeRESUMO
The human T-cell lymphotropic virus type I (HTLV-1) Tax transactivator initiates transformation in adult T-cell leukemia/lymphoma (ATL), a highly aggressive chemotherapy-resistant malignancy. The arsenic/interferon combination, which triggers degradation of the Tax oncoprotein, selectively induces apoptosis of ATL cell lines and has significant clinical activity in Tax-driven murine ATL or human patients. However, the role of Tax loss in ATL response is disputed, and the molecular mechanisms driving degradation remain elusive. Here we demonstrate that ATL-derived or HTLV-1-transformed cells are dependent on continuous Tax expression, suggesting that Tax degradation underlies clinical responses to the arsenic/interferon combination. The latter enforces promyelocytic leukemia protein (PML) nuclear body (NB) formation and partner protein recruitment. In arsenic/interferon-treated HTLV-1 transformed or ATL cells, Tax is recruited onto NBs and undergoes PML-dependent hyper-sumoylation by small ubiquitin-like modifier (SUMO)2/3 but not SUMO1, ubiquitination by RNF4, and proteasome-dependent degradation. Thus, the arsenic/interferon combination clears ATL through degradation of its Tax driver, and this regimen could have broader therapeutic value by promoting degradation of other pathogenic sumoylated proteins.
Assuntos
Arsenicais/farmacologia , Produtos do Gene tax/metabolismo , Interferons/farmacologia , Leucemia-Linfoma de Células T do Adulto/virologia , Proteínas Nucleares/metabolismo , Proteólise/efeitos dos fármacos , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Transformação Celular Viral/efeitos dos fármacos , Quimioterapia Combinada , Citometria de Fluxo , Imunofluorescência , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Imunoprecipitação , Leucemia-Linfoma de Células T do Adulto/genética , Proteína da Leucemia Promielocítica , Reagentes de Sulfidrila/farmacologia , Sumoilação/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.
Assuntos
Apoptose/fisiologia , Membranas Mitocondriais/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Cisteína/química , Humanos , Camundongos , Membranas Mitocondriais/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estilbenos , Reagentes de Sulfidrila , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Arsenic is naturally occurring toxic metalloid and drinking As2 O3 containing water are recognized to be related to increased risk of neurotoxicity, liver injury, blackfoot disease, hypertension, and cancer. On the contrary, As2 O3 has been an ancient drug used in traditional Chinese medicine with substantial anticancer activities, especially in the treatment of acute promyelocytic leukemia as well as chronic wound healing. However, the cytotoxicity and detail mechanisms of As2 O3 action in solid cancer cells, such as oral cancer cells, are largely unknown. In this study, we have primarily cultured four pairs of tumor and nontumor cells from the oral cancer patients and treated the cells with As2 O3 alone or combined with dithiothreitol (DTT). The results showed that 0.5 µM As2 O3 plus 20 µM DTT caused a significant cell death of oral cancer cells but not the nontumor cells. Also As2 O3 plus DTT upregulated Bax and Bak, downregulated Bcl-2 and p53, caused a loss of mitochondria membrane potential in oral cancer cells. On the other way, As2 O3 also triggered endoplasmic reticulum stress and increased the levels of glucose-regulated protein 78, calpain 1 and 2. Our results suggest that DTT could synergistically enhance the effects of As2 O3 on killing oral cancer cells while nontoxic to the nontumor cells. The combination is promising for clinical practice in oral cancer therapy and worth further investigations. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 17-27, 2017.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Ditiotreitol/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Doenças Mitocondriais/induzido quimicamente , Neoplasias Bucais/tratamento farmacológico , Óxidos/toxicidade , Reagentes de Sulfidrila/farmacologia , Trióxido de Arsênio , Arsenicais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Sinergismo Farmacológico , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Neoplasias Bucais/patologiaRESUMO
Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study, C. jejuniâ arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III) > Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus, ArsP is the first identified efflux system specific for trivalent organoarsenicals.
Assuntos
Arsenitos/metabolismo , Campylobacter jejuni/enzimologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Arseniatos/metabolismo , Arsenicais/metabolismo , Arsenicais/farmacologia , Arsenitos/farmacologia , Campylobacter jejuni/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/metabolismo , Roxarsona/química , Roxarsona/farmacologia , Reagentes de Sulfidrila/metabolismoRESUMO
Water-soluble trialkylphosphines such as tris(carboxyethyl)phosphine (TCEP) and trishydroxypropyl phosphine (THPP) are effective agents for reducing disulfide bonds in proteins and are increasingly becoming the reagents of choice for bioconjugation strategies that modify cysteine (thiol containing) amino acids. These reducing agents are often considered as being chemically compatible with Michael acceptors such as maleimides and, as such, are often not removed prior to performing protein conjugation reactions. Here, we demonstrate the rapid and irreversible reaction of both TCEP and THPP with derivatives of the commonly employed thiol alkylating groups, maleimide and vinyl sulfone. Mechanistic investigations revealed distinct differences between the reactions of TCEP and THPP with maleimide, leading to the production of either nonproductive ylenes or succidimidyl derivatives, respectively. Importantly, we also demonstrate the incorporation of nonproductive ylenes formed between maleimide and TCEP into the Pneumococcal capsular polysaccharide Pn6b following strategies employed toward the production of conjugate vaccines.