RESUMO
Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , RNA Interferente Pequeno/farmacologia , Ubiquitinação/efeitos dos fármacos , Benzamidas , Proteínas de Fusão bcr-abl/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Lisossomos/genética , Lisossomos/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Ubiquitinação/genéticaRESUMO
Gold nanoparticles (AuNP) have potential applications in drug delivery, cancer diagnosis and therapy, food industry and environment remediation. However, little is known about their potential toxicity or fate in the environment. Mytilus edulis was exposed in tanks to 750 ppb AuNP (average diameter 5.3 ± 1 nm) for 24h to study in vivo biological effects of nanoparticles. Traditional biomarkers and an affinity procedure selective for thiol-containing proteins followed by two-dimensional electrophoresis (2DE) separations were used to study toxicity and oxidative stress responses. Results were compared to those obtained for treatment with cadmium chloride, a well known pro-oxidant. M. edulis mainly accumulated AuNP in digestive gland which also showed higher lipid peroxidation. One-dimensional SDS/PAGE (1DE) and 2DE analysis of digestive gland samples revealed decreased thiol-containing proteins for AuNP. Lysosomal membrane stability measured in haemolymph gave lower values for neutral red retention time (NRRT) in both treatments but was greater in AuNP. Oxidative stress occurred within 24h of AuNP exposure in M. edulis. Previously we showed that larger diameter AuNP caused modest effects, indicating that nanoparticle size is a key factor in biological responses to nanoparticles. This study suggests that M. edulis is a suitable model animal for environmental toxicology studies of nanoparticles.
Assuntos
Ouro/toxicidade , Nanopartículas Metálicas/toxicidade , Mytilus edulis/fisiologia , Estresse Oxidativo , Poluentes Químicos da Água/toxicidade , Animais , Peroxidação de Lipídeos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Malondialdeído/metabolismo , Nanopartículas Metálicas/ultraestrutura , Vermelho Neutro/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Protein thiols contribute significantly to antioxidant defence and selective oxidation of cysteines is important in signal transduction even in sub-stress scenarios. However, cysteine is the second rarest residue in proteins and it can be difficult to target low-abundance thiol (-SH)-containing proteins in proteomic separations. Activated thiol sepharose (ATS) allows covalent selection of -SH-containing proteins which can then be recovered by reduction with mercaptoethanol or dithiothreitol. This is a robust method for enriching -SH-containing proteins. We have used ATS to estimate the percentage (by weight) of thiol-containing proteins in cell extracts from a range of biological sources: a bacterium, Escherichia coli; a fungus, Trichoderma harzianum; and a bivalve mollusc Mytilus edulis. -SH-containing proteins account for 2.52% (E. coli), 1.4% (T. harzianum) and 1.4% (M. edulis) of total protein. Exposure to pro-oxidants did not materially alter these values. On removal of low M(r) thiols such as glutathione, the values for M. edulis did not significantly change but those for T. harzianum increased threefold. The two-dimensional electrophoresis profiles of ATS-selected proteins for each organism were compared in control and pro-oxidant-exposed preparations. This revealed that some proteins present in controls were absent in pro-oxidant-treated extracts which we attribute to thiol oxidation. ATS has significant potential in enrichment for -SH-containing proteins in redox proteomics.
Assuntos
Oxirredução , Estresse Oxidativo , Proteoma/química , Proteômica/métodos , Compostos de Sulfidrila/química , Animais , Cisteína/metabolismo , Dissulfetos/metabolismo , Glutationa/metabolismo , Espécies Reativas de Oxigênio , Sefarose/análogos & derivados , Sefarose/química , Compostos de Sulfidrila/metabolismoRESUMO
If reactive oxygen species (ROS) levels exceed antioxidant defences, oxidative stress occurs; a common response to environmental pollutants. Proteins absorb â¼70% of ROS, altering amino acid side-chains. Cys (-SH) oxidises to sulphenic (-SOH), sulphinic (-SO(2)H), cysteic (-SO(3)H) acids and disulphide bridges (-S-S-). Two-dimensional electrophoresis (2DE) under-selects certain protein categories (e.g. extreme pI, small proteins) so activated thiol sepharose (ATS) was used to select sub-proteomes of thiol-containing proteins in menadione-exposed Escherichia coli. ATS bound thiol-containing proteins (but not oxidised thiols) via mixed disulphides. Tryptic digestion of bead-bound proteins was followed by LC-tandem MS. Many proteins were identified in controls with significantly fewer in menadione-treated cells (e.g. chaperonins, transcription/translation-related and ribosomal proteins; aminoacyl tRNA synthetases and metabolic enzymes. Non-denaturing ATS capture (followed by reduction) demonstrated lower specific activities of key enzymes which is attributed to thiol oxidation. This method may be generally useful in ecotoxicology for identification of oxidative stress targets.
Assuntos
Ecotoxicologia/métodos , Estresse Oxidativo , Proteínas/metabolismo , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Escherichia coli , Ligação Proteica , Proteômica , Espécies Reativas de Oxigênio , Sefarose/análogos & derivados , Vitamina K 3RESUMO
Activated thiol-Sepharose (ATS) facilitates selection of thiol-containing proteins. In control- and menadione-treated Escherichia coli, batch selection performed under denaturing conditions revealed distinct two-dimensional electrophoresis (2DE) patterns. Using shotgun proteomics, 183 thiol-containing proteins were identified in control ATS-selected extracts and 126 were identified in menadione-treated E. coli, with 85 proteins being common to both. More than 90% of identified proteins contained one or more cysteines. Blocking with N-ethyl maleimide followed by reduction facilitated ATS-based selection of disulfide-containing proteins. In total, 62 proteins were unique to control cells and 164 were identified in menadione-treated E. coli cells, with 29 proteins being common to both. Proteins from menadione-treated cells were excised from 2DE gels, digested with trypsin, and identified by peptide mass fingerprinting. This revealed 19 unique proteins, 14 of which were identified by shotgun proteomics. Outer membrane proteins A, C, W, and X and 30S ribosomal protein S1 were found in 2DE but not by shotgun proteomics. Foldases, ribosomal proteins, aminoacyl transfer RNA (tRNA) synthetases, and metabolic and antioxidant enzymes were prominent among identified proteins, and many had previously been found to respond to, and be targets for, oxidative stress in E. coli. ATS provides a convenient and rapid way to select thiol-containing proteins.
Assuntos
Dissulfetos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli , Sefarose , Compostos de Sulfidrila , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/análise , Oxidantes/farmacologia , Proteômica , Vitamina K 3/farmacologiaRESUMO
Relatively little is known about how gold nanoparticles (GNP) might interact in vivo with marine organisms. Mytilus edulis was exposed (24h) to approximately 15 nm GNP, menadione and both compounds simultaneously (GNP/menadione). GNP was detected by inductively coupled plasma-optical emission spectroscopy mainly in digestive gland of samples exposed to GNP though not GNP/menadione, perhaps due to impaired feeding. Thioredoxin reductase activity and malondialdehyde levels were determined in all tissues. Thioredoxin reductase inhibition was detected only in digestive gland exposed to menadione whilst malondialdehyde levels did not vary in response to treatment in all tissues. GNP caused a decrease in the reduced/oxidized glutathione ratio in digestive gland, but no difference was found in other tissues or for other treatments. One dimensional electrophoresis of proteins containing thiol groups was performed in all tissues and revealed a reduction in protein thiols for all treatments in digestive gland. Two dimensional electrophoresis of digestive gland extracts, from GNP and control groups, showed decreased levels of thiol proteins in response to GNP which we attribute to oxidation. Our results suggest that GNP causes a modest level of oxidative stress sufficient to oxidize thiols in glutathione and proteins but without causing lipid peroxidation or induction of thioredoxin reductase activity.
Assuntos
Ouro/farmacologia , Mytilus edulis/efeitos dos fármacos , Vitamina K 3/farmacologia , Animais , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/metabolismo , Eletroforese em Gel Bidimensional , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Ouro/farmacocinética , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Mytilus edulis/metabolismo , Oxirredução/efeitos dos fármacos , Espectrofotometria , Compostos de Sulfidrila/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Vitaminas/farmacologiaRESUMO
Pro-oxidant effects of extremely low frequency (ELF) 50-Hz magnetic fields were investigated in the land snail Helix aspersa exposed both in short-term laboratory treatments and under field conditions by maintaining the organisms in the proximity of a power line for up to 2 months. Oxidative perturbations were investigated as individual antioxidants (catalase, glutathione reductase, glutathione S-transferases, and total glutathione) and total scavenging capacity toward peroxyl radicals and hydroxyl radicals. Accumulation of lipid peroxidation products, destabilization of lysosomal membranes, and loss of DNA integrity were also evaluated as markers of cell damage. The overall results indicated an oxidative challenge caused by ELF magnetic fields with particularly prompt and sensitive responses for catalase, glutathione reductase, and the overall capability to neutralize peroxyl radicals. Cell injuries occurred to different extents according to duration and intensity of electromagnetic exposure and confirmed complex cause-effect relationships between pro-oxidant factors, efficiency of antioxidant defenses, and the onset of oxidative toxicity. This study highlights the importance of a multimarker approach for detecting a wide panel of biological responses, the necessity of investigating the long-term effects of early oxidative responses, and the role of ELF in enhancing susceptibility to other forms of pathologies or diseases.