RESUMO
The nuclear transport receptor importin-ß/karyopherin-ß1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-ß regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-ß's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-ß form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-ß affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-ß interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-ß, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-ß's nucleoporin-binding residues. Concomitantly, importin-ß overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-ß function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Taxoides/farmacologia , beta Carioferinas/química , beta Carioferinas/metabolismo , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Paclitaxel/farmacologia , Ligação ProteicaRESUMO
Among natural products under investigation for their additive potential in cancer prevention and treatment, the flavonoid quercetin has received attention for its effects on the cell cycle arrest and apoptosis. In the past, we addressed this issue in K562 cells, a cellular model of the human chronic myeloid leukemia. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) proteomics with the aim to increase knowledge on the regulative and metabolic pathways modulated by quercetin in these cells. After 24 h of quercetin treatment, we observed that apoptosis was not completely established, thus we selected this time range to capture quantitative data. As a result, we were able to achieve a robust identification of 1703 proteins, and to measure fold changes between quercetin-treated and untreated cells for 1206 proteins. Through a bioinformatics functional analysis on a subset of 112 proteins, we propose that the apoptotic phenotype of K562 cells entails a significant modulation of the translational machinery, RNA metabolism, antioxidant defense systems, and enzymes involved in lipid metabolism. Finally, we selected eight differentially expressed proteins, validated their modulated expression in quercetin-treated K562 cells, and discussed their possible role in flavonoid cytotoxicity. This quantitative profiling, performed for the first time on this type of tumor cells upon treatment with a flavonoid, will contribute to revealing the molecular basis of the multiplicity of the effects selectively exerted by quercetin on K562 cells.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteoma/efeitos dos fármacos , Proteômica/métodos , Quercetina/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Marcação por Isótopo , Células K562 , Metabolismo dos Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fatores de TempoRESUMO
Amyloid-treated microglia prime and sustain neuroinflammatory processes in the central nervous system activating different signalling pathways inside the cells. Since a key role for PARP-1 has been demonstrated in inflammation and in neurodegeneration, we investigated PARylated proteins in resting and in ß-amyloid peptide treated BV2 microglial cells. A total of 1158 proteins were identified by mass spectrometry with 117 specifically modified in the amyloid-treated cells. Intervention of PARylation on the proteome of microglia showed to be widespread in different cellular districts and to affect various cellular pathways, highlighting the role of this dynamic post-translational modification in cellular regulation. Ubiquitination is one of the more enriched pathways, encompassing PARylated proteins like NEDD4, an E3 ubiquitine ligase and USP10, a de-ubiquitinase, both associated with intracellular responses induced by ß-amyloid peptide challenge. PARylation of NEDD4 may be involved in the recruiting of this protein to the plasma membrane where it regulates the endocytosis of AMPA receptors, whereas USP10 may be responsible for the increase of p53 levels in amyloid stimulated microglia. Unfolded protein response and Endoplasmic Reticulum Stress pathways, strictly correlated with the Ubiquitination process, also showed enrichment in PARylated proteins. PARylation may thus represent one of the molecular switches responsible for the transition of microglia towards the inflammatory microglia phenotype, a pivotal player in brain diseases including neurodegenerative processes. The establishment of trials with PARP inhibitors to test their efficacy in the containment of neurodegenerative diseases may be envisaged.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Sobrevivência Celular/fisiologia , Microglia/metabolismo , Fragmentos de Peptídeos/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacosRESUMO
In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Microglia/metabolismo , Proteômica/métodos , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Marcação por Isótopo , Camundongos , Microglia/efeitos dos fármacos , RatosRESUMO
Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary's bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Extratos Vegetais/farmacologia , Rosmarinus/química , Abietanos/farmacologia , Apigenina/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaios de Seleção de Medicamentos Antitumorais , Glucuronatos/farmacologia , Humanos , Luteolina/farmacologia , Melanoma/tratamento farmacológico , Estresse Oxidativo , Carbonilação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Microglia are resident macrophages in the central nervous system, whose participation against exogenous injuries and infections is mainly marked by an immediate release of inflammatory cytokines along with a toxic efflux of superoxide radicals. Indeed, many lines of evidence indicate that persistent activation of these cells turns their neuroprotective phenotype into a neurotoxic one, which contributes to destroy neuronal activity and induces neuronal loss in several neurodegeneration processes, such as Alzheimer's disease. In this study we attempted to fill-in the gap in our knowledge about redox regulation of amyloid activated microglia. With this aim, we carried out a robust and comprehensive characterization of the reversibly redox modified proteome both at the level of resting and amyloid-activated BV2 cells, an immortalised cell line of murine microglia. The approach we used combined the selective enrichment of reversible redox modified proteins through a biotin bait with nanoscale liquid chromatography tandem mass spectrometry of their proteolytic peptides. By this reliable approach, we identified 60 proteins changing the redox status of their selective cysteine residues upon treatment with the amyloidogenic Aß25-35 peptide. These results assessed that in microglia stimulated by amyloids, redox modifications of the proteome specifically target proteins involved in crucial cell processes, i.e. those involved in the protein synthesis. In particular, for peroxiredoxin-6 (Prdx6) and Ras-related C3 botulinum toxin substrate 1 (Rac1) we suggest mechanisms through which reversible redox modifications could affect the peculiar role of microglia in amyloidogenic injury, which at the same time reinforce the oxidative burst and resist toward it. Moreover, the redox modulation we observed on chloride intracellular channel protein 1 (CLIC1) strengthens the structural and functional relationship between the oxidative stress and the metamorphic transition of this protein from a soluble form to an integral membrane form. The redox signatures we determined might also provide neurologists with more specific and reliable biomarkers to distinguish the diverse microglia status in neurodegeneration and then to drive targeted drug design.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Microglia/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteoma/análise , Proteoma/efeitos dos fármacos , Animais , Linhagem Celular , Camundongos , Microglia/metabolismo , Oxirredução , Proteoma/química , ProteômicaRESUMO
Microglia are macrophages within the central nervous system playing a central role in neurodegenerative disorders. Although the initial engagement of microglia seems to be neuroprotective, many lines of evidence indicate that its persistent activation contributes to dismantle neuronal activity and to induce neuronal loss. The molecular pathways that lead from amyloid interaction with membrane receptors to the microglial activation have been extensively investigated, although a definitive picture is not yet at hand. In this work, primary and immortalized microglial cells were treated with a synthetic form of Aß peptides, and relative abundance of acetylated and phosphorylated STAT3 were assayed. Results highlight, for the first time, three distinctive sequential events: i) an earlier event marked by the increase in the level of STAT3 acetylated species, followed by ii) a later increase in the level of STAT3 phosphorylated form, and finally iii) an involvement of phosphorylated STAT3 in the increase in expression of the 14-3-3 epsilon, a protein frequently associated with neurodegenerative diseases and known to be a marker of Aß-activated microglia. These data outline a complex, time-dependent modification of STAT3 signalling triggered by amyloid in the microglial compartments, that once confirmed by in vivo experiments will broaden the knowledge of the molecular basis of amyloid neurotoxicity.
Assuntos
Peptídeos beta-Amiloides/fisiologia , Microglia/metabolismo , Fator de Transcrição STAT3/metabolismo , Acetilação , Animais , Linhagem Celular , Camundongos , Microglia/citologia , FosforilaçãoRESUMO
The development of plant root systems is characterized by a high plasticity, made possible by the continual propagation of new meristems. Root architecture is fundamental for overall plant growth, abiotic stress resistance, nutrient uptake, and response to environmental changes. Understanding the function of genes and proteins that control root architecture and stress resistance will contribute to the development of more sustainable systems of intensified crop production. To meet these challenges, proteomics provide the genome-wide scale characterization of protein expression pattern, subcellular localization, post-translational modifications, activity regulation, and molecular interactions. In this review, we describe a variety of proteomic strategies that have been applied to study the proteome of the whole organ and of specific cell types during root development. Each has advantages and limitations, but collectively they are providing important insights into the mechanisms by which auxin structures and patterns the root system and into the interplay between signaling networks, auxin transport and growth. The acquisition of proteomic, transcriptomic, and metabolomic data sets of the root apex on the cell scale has revealed the high spatial complexity of regulatory networks and fosters the use of new powerful proteomic tools for a full understanding of the control of root developmental processes and environmental responses.
Assuntos
Arabidopsis/genética , Biomarcadores/metabolismo , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Proteômica/métodos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Cromatografia Líquida , Perfilação da Expressão Gênica , Ácidos Indolacéticos/química , Estrutura Molecular , Raízes de Plantas/metabolismo , Espectrometria de Massas em TandemRESUMO
Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Several evidences suggest that MS can be considered a multi-factorial disease in which both genetics and environmental factors are involved. Among proposed candidates, growing results support the involvement of oxidative stress (OS) in MS pathology. The aim of this study was to investigate the role of OS in event of exacerbations in MS on serum of relapsing-remitting (RR-MS) patients, either in relapsing or remitting phase, with respect to serum from healthy subjects. We applied proteomics and redox proteomics approaches to identify differently expressed and oxidatively modified proteins in the low-abundant serum protein fraction. Among differently expressed proteins ceruloplasmin, antithrombin III, clusterin, apolipoprotein E, and complement C3, were up-regulated in MS patients compared with healthy controls. Further by redox proteomics, vitamin D-binding protein showed a progressive trend of oxidation from remission to relapse, respect with controls. Similarly, the increase of oxidation of apolipoprotein A-IV confirmed that levels of OS are elevated with the progression of the disease. Our findings support the involvement of OS in MS and suggest that dysfunction of target proteins occurs upon oxidative damage and correlates with the pathology.
Assuntos
Proteínas Sanguíneas/metabolismo , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/patologia , Estresse Oxidativo , Proteômica/métodos , Adulto , Apolipoproteínas A/metabolismo , Estudos de Casos e Controles , Demografia , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/metabolismo , Oxirredução , Carbonilação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína de Ligação a Vitamina D/metabolismoRESUMO
The pathological form of prion protein (PrP(Sc)), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc) extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP90â231) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc). In the present study we demonstrate that hPrP90â231, pre-incubated with 10 mM Caâºâº and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP90â231 bearing pathogenic mutations (D202N and E200K). We also report that Caâºâº binding to hPrP90â231 induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP90â231 cytotoxicity. Finally, by in silico structural analysis, we propose that Caâºâº binding to hPrP90â231 modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.
Assuntos
Cálcio/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Príons/química , Príons/toxicidade , Estrutura Secundária de Proteína/efeitos dos fármacos , Aminoácidos/genética , Animais , Sítios de Ligação , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Cricetinae , Endopeptidase K/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Bicamadas Lipídicas/química , Potenciais da Membrana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/genética , Príons/genética , Desnaturação ProteicaRESUMO
Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization.
Assuntos
Proteínas de Transporte/metabolismo , Citocinese , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Divisão Celular , Linhagem Celular , Linhagem Celular Tumoral , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Histonas/genética , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , TetraploidiaRESUMO
Highly stable natural scaffolds which tolerate multiple amino acid substitutions represent the ideal starting point for the application of rational redesign strategies to develop new catalysts of potential biomedical and biotechnological interest. The knottins family of disulphide-constrained peptides display the desired characteristics, being highly stable and characterized by hypervariability of the inter-cysteine loops. The potential of knottins as scaffolds for the design of novel copper-based biocatalysts has been tested by engineering a metal binding site on two different variants of an ω-conotoxin, a neurotoxic peptide belonging to the knottins family. The binding site has been designed by computational modelling and the redesigned peptides have been synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupricyclin-1 and -2, bind one Cu(2+) ion per molecule with nanomolar affinity. Cupricyclins display redox activity and catalyze the dismutation of superoxide anions with an activity comparable to that of non-peptidic superoxide dismutase mimics. We thus propose knottins as a novel scaffold for the design of catalytically-active mini metalloproteins.
Assuntos
Conotoxinas/química , Metaloproteínas/química , Peptídeos/química , Sítios de Ligação , Bloqueadores dos Canais de Cálcio , Cobre/metabolismo , Desenho de Fármacos , Metaloproteínas/síntese química , Metaloproteínas/genética , Neurotoxinas , Oxirredução , Engenharia de ProteínasRESUMO
Microglia-mediated inflammation in the central nervous system is a hallmark of the pathogenesis of several neurodegenerative diseases including Alzheimer's disease. Microglial cells activation follows the deposition of amyloid ß fibrils and it is generally considered a triggering factor in the early steps of the onset of Alzheimer's disease. Although the initial engagement of microglia seems to play a neuroprotective role, many lines of evidence indicate that a persistent activation with the production of proinflammatory molecules contributes to dismantle neuronal activity and to induce neuronal loss occurring in neurodegenerative diseases. To date, limited proteomic data are available on activated microglial cells in response to extracellular amyloidogenic peptides. In this study, murine microglial cells have been employed to investigate the effects of amyloid ß peptides in triggering microglial activation. The response was monitored at the proteome level through a two-dimensional gel electrophoresis-based approach. Results show only a limited number of differentially expressed proteins, among these a more acidic species of the cytosolic actin, and the 14-3-3ε protein, found significantly upregulated in Aß-activated cells. 14-3-3ε belongs to a regulatory protein family involved in important cellular processes, including those leading to neurodegenerative diseases, and thus its increased expression suggests a role of this protein in tuning microglia activation.
Assuntos
Proteínas 14-3-3/metabolismo , Inflamação/metabolismo , Microglia/patologia , Proteínas 14-3-3/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Proteínas Amiloidogênicas , Animais , Biomarcadores/metabolismo , Linhagem Celular Transformada , Inflamação/induzido quimicamente , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteoma/genética , Proteoma/metabolismo , Regulação para CimaRESUMO
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative pathologies characterized by the accumulation of amyloid fibrils mainly composed of the pathological isoform of the prion protein (PrP(TSE)). PrP(TSE) pre-amyloid fibrils are supposed to induce neurodegenerative lesions possibly through the alteration of membrane permeability. The effect of PrP(TSE) on cellular membranes has been modeled in vitro by synthetic peptides that are, however, only partially representative of PrP(TSE) isoforms found in vivo. In the present work we show that a synthetic membrane exposed to PrP27-30 extracted from TSE-infected hamster brains changes its permeability because of the formation of molecular pores that alter the conductance of the synthetic lipid bilayer. Synthetic membrane challenged with the recombinant prion peptide PrP90-231 shows a much lower conductance. Elevation of calcium ion concentration not only increases the current amplitude due to the action of both PrP27-30 and PrP90-231 on the membrane, but also amplifies the interaction of PrP90-231 with the lipid bilayer.
Assuntos
Bicamadas Lipídicas , Príons/fisiologia , Animais , Cricetinae , Transporte de Íons , MesocricetusRESUMO
PURPOSE: The present study aims to evaluate a set of oxidative stress biomarkers in the amniotic fluid (AF) of women carrying Down syndrome (DS) fetuses that could prove in vivo the early occurrence of oxidative damage in DS. EXPERIMENTAL DESIGN: To assess the extent of protein oxidation in DS AF, we measured protein carbonylation and protein-bound HNE by slot-blot analysis, total and oxidized GSH levels by enzymatic assay and heat shock proteins (HSPs) thioredoxin (Trx) induction by Western blot. Further, by a redox proteomics approach specific targets of protein carbonylation were identified. RESULTS: We found increased levels of oxidative stress, as indexed by increased protein oxidation, lipid peroxidation, reduction of GSH and Trx levels and induction of the HSP response. By a redox proteomics approach, we identified selective proteins which showed increased oxidation in DS fetuses compared with healthy controls. The identified proteins are involved in iron homeostasis (ceruloplasmin and transferin), lipid metabolism (zinc-α2-glycoprotein, retinol-binding protein 4 and apolipoprotein A1) and inflammation (complement C9, α-1B-glycoprotein, collagen α-1V chain) with critical relevance in the clinical outcome of DS. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that oxidative damage is an early event in the DS pathogenesis and might contribute to the development of deleterious DS phenotypes, including abnormal development and AD-like neuropathology.
Assuntos
Líquido Amniótico/química , Síndrome de Down/metabolismo , Estresse Oxidativo , Primeiro Trimestre da Gravidez/metabolismo , Proteínas/análise , Proteômica , Adulto , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Feminino , Humanos , Oxirredução , Estresse Oxidativo/genética , Fenótipo , Gravidez , Primeiro Trimestre da Gravidez/genética , Proteínas/genética , Proteínas/metabolismo , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Human Papillomavirus (HPV)-16 is a paradigm for "high-risk" HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation. METHODOLOGY/PRINCIPAL FINDINGS: By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7. CONCLUSIONS/SIGNIFICANCE: This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells.
Assuntos
Glutationa S-Transferase pi/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Transformação Celular Neoplásica , Regulação Viral da Expressão Gênica , Humanos , Queratinócitos/virologia , Espectrometria de Massas/métodos , Conformação Molecular , Mutação , Proteínas E7 de Papillomavirus , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Repressoras/metabolismoRESUMO
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation in the CNS of a pathological conformer (PrP(TSE)) of the host-encoded cellular prion protein (PrP(C)). PrP(TSE) has a central role in the pathogenesis of the disease but other factors are likely involved in the pathological process. In this work we employed a multi-step proteomic approach for the identification of proteins that co-purify with the protease-resistant core of PrP(TSE) (PrP27-30) extracted from brains of hamsters with experimental scrapie. We identified ferritin, calcium/calmodulin-dependent protein kinase alpha type II, apolipoprotein E, and tubulin as the major components associated with PrP27-30 but also trace amounts of actin, cofilin, Hsp90alpha, the gamma subunit of the T-complex protein 1, glyceraldehyde 3-phosphate dehydrogenase, histones, and keratins. Whereas some of these proteins (tubulin and ferritin) are known to bind PrP, other proteins (calcium/calmodulin-dependent protein kinase alpha type II, Hsp90alpha) may associate with PrP(TSE) fibrils during disease. Apolipoprotein E and actin have been previously observed in association with PrP(TSE), whereas cofilin and actin were shown to form abnormal rods in the brain of patients with Alzheimer disease. The roles of these proteins in the development of brain lesions are still unclear and further work is needed to explain their involvement in the pathogenesis of TSEs.
Assuntos
Encéfalo/patologia , Proteína PrP 27-30/metabolismo , Proteínas/metabolismo , Proteômica , Scrapie/metabolismo , Animais , Apolipoproteínas E/análise , Apolipoproteínas E/metabolismo , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/análise , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cricetinae , Proteína PrP 27-30/análise , Proteína PrP 27-30/isolamento & purificação , Proteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Increasing evidence supports the role of oxidative stress in cancer development. Ultraviolet (UV) irradiation is one of the major sources of oxidative stress through the generation of reactive oxygen species (ROS). Besides the physiological function of ROS in cellular homeostasis, accumulating reports suggest that ROS are involved in all stages of multistep cancer development. In order to investigate the involvement of oxidative damage into the mechanisms of tumour progression, we used a parallel proteomic approach to analyse the protein expression profile and to identify oxidatively modified proteins in human papillomavirus (HPV)-transformed keratinocytes (HK-168 cells) upon ultraviolet B (UVB) exposure. The HK-168 cells were obtained from normal human epidermal keratinocytes transfected with the whole genome of the high-risk HPV type 16, unanimously recognized as an etiological agent of cervical carcinoma. Because of its year-long latency, this tumour offers a convenient model to study the role of environmental concurring agents in the multistep malignant progression. By the protein expression profile, we identified 21 proteins that showed different expression levels in HK-168 cells treated with UVB in comparison with untreated cells. Focusing on the oxidative modifications occurring at the protein level, we identified five proteins that showed elevated protein carbonyls levels: alpha-enolase, heat shock protein 75, annexin 2, elongation factor Tu and elongation factor gamma. Our results indicate that UVB-induced oxidative stress perturbs the normal redox balance and shifts HPV-transformed keratinocytes into a state in which the carbonylation of specific proteins is systematically induced. We suggest that UVB-induced modulation of protein expression combined with oxidative modification lead to protein dysfunction that might contribute to the malignant progression of transformed cells.
Assuntos
Queratinócitos/efeitos da radiação , Papillomaviridae/fisiologia , Proteínas/metabolismo , Proteômica , Raios Ultravioleta , Western Blotting , Linhagem Celular Transformada , Eletroforese em Gel Bidimensional , Humanos , Queratinócitos/virologia , Oxirredução , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Contryphans are bioactive peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post-translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues, and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well-defined structure in solution, conserved in all members of the family, and tolerant to multiple substitutions. The potential of Contryphans as scaffolds for the design of redox-active (macro)molecules was tested by engineering a copper-binding site on two different variants of the natural peptide Contryphan-Vn. The binding site was designed by computational modeling, and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance, and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg-Cupryphan, bind Cu(2+) ions with a 1:1 stoichiometry and a K(d) in the 100 nM range. Other divalent metals (e.g., Zn(2+) and Mg(2+)) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other nonpeptidic superoxide dismutase mimics. We conclude that the Contryphan motif represents a natural robust scaffold which can be engineered to perform different functions, providing additional means for the design of catalytically active mini metalloproteins.
Assuntos
Cátions Bivalentes/metabolismo , Caramujo Conus/genética , Cobre/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Biologia Computacional , Metaloproteínas/genética , Metaloproteínas/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Peptídeos/síntese química , Peptídeos/genética , Engenharia de Proteínas , Superóxido Dismutase/metabolismoRESUMO
In the melanoma M14 cell line, we found that the antimetastatic protein NM23/nucleoside diphosphate kinase binds to the promoters of the oncogene cMYC and of P53, a gene often mutated in human cancer (Cervoni et al. [2006] J. Cell. Biochem. 98:421-428). In a further study, we find now that IFI16, a transcriptional repressor, in both promoters binds to the G-rich fragment that also binds NM23/NDPK. These fragments possess non-B DNA structures. Moreover, by sequential chromatin immunoprecipitation (re-ChIP) we show that the two proteins (IFI16 and NM23/NDPK) are simultaneously bound in vivo to the same DNA fragments. Since P53 stimulates apoptosis and inhibits cellular growth, and cMYC promotes cell growth and, in several instances, also apoptosis, the presence of NM23 and IFI16 on the same DNA fragments suggests their common involvement in the reduced development of some tumors.