RESUMO
Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic responses dependent upon cellular context.
Assuntos
Antagonistas de Androgênios/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/isolamento & purificação , Próstata/efeitos dos fármacos , Proteoma/isolamento & purificação , Receptores Androgênicos/química , Sequência de Aminoácidos , Antagonistas de Androgênios/química , Anilidas/química , Anilidas/farmacologia , Linhagem Celular Tumoral , Acetato de Ciproterona/química , Acetato de Ciproterona/farmacologia , Flutamida/análogos & derivados , Flutamida/química , Flutamida/farmacologia , Humanos , Masculino , Anotação de Sequência Molecular , Dados de Sequência Molecular , Mutação , Nandrolona/análogos & derivados , Nandrolona/química , Nandrolona/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Nitrilas/química , Nitrilas/farmacologia , Próstata/metabolismo , Próstata/patologia , Proteoma/genética , Proteoma/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais , Compostos de Tosil/química , Compostos de Tosil/farmacologiaRESUMO
Smooth muscle cells (SMC) contribute to the development and stability of atherosclerotic lesions. The molecular mechanisms that mediate their properties are incompletely defined. We employed proteomics and in vitro functional assays to identify the unique characteristics of intimal SMC isolated from human carotid endarterectomy specimens and medial SMC from thoracic aortas and carotids. We verified our findings in the Tampere Vascular Study. Human atheroma-derived SMC exhibit decreased expression of mitochondrial proteins ATP Synthase subunit-beta and Aldehyde dehydrogenase 2, and decreased mitochondrial activity when compared to control SMC. Moreover, a comparison between plaque-derived SMC isolated from patients with or without recent acute cerebrovascular symptoms uncovered an increase in Annexin A1, an endogenous anti-inflammatory protein, in the asymptomatic group. The deletion of Annexin A1 or the blockade of its signaling in SMC resulted in increased cytokine production at baseline and after stimulation with the pro-inflammatory cytokine Tumor Necrosis Factor α. In summary, our proteomics and biochemical analysis revealed mitochondrial damage in human plaque-derived SMC as well as a role of Annexin A1 in reducing the production of pro-inflammatory mediators in SMC.
Assuntos
Anexina A1/metabolismo , Aterosclerose/patologia , Doenças das Artérias Carótidas/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteoma/metabolismo , Adulto , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial , Animais , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Citocinas/metabolismo , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Músculo Liso Vascular/patologia , Oxirredução , Peroxirredoxinas/metabolismo , Fenótipo , Análise de Componente Principal , ProteômicaRESUMO
BACKGROUND: Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly IratioC). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-kappaB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.
Assuntos
Citocinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas/metabolismo , Receptores Toll-Like/metabolismo , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteínas/genética , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Espectrometria de Massas em Tandem , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/genética , Fator de Necrose Tumoral alfaRESUMO
We describe the characterization of polyclonal antibodies directed against the whole mitochondrial subproteome, as obtained by hyperimmunization of rabbits with an organelle fraction purified from human skeletal muscle and lysed by sonication. After 2-DE separations with either blue native electrophoresis or IPG as first dimension and blotting, the polyspecific antibodies detect 113 proteins in human muscle mitochondria, representative of all major biochemical pathways and oxidative phosphorylation (OXPHOS) complexes, and cross-react with 28 proteins in rat heart mitochondria. Using as sample cryosections of human muscle biopsies lysed in urea/thiourea/CHAPS, the mitochondrial subproteome can be detected against the background of contractile proteins. When comparing with controls samples from mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes patients, immunoblotting shows in the latter a drastic reduction for the subunits of OXPHOS complex I as well as an increase of several enzymes, including ATP synthase. This finding is the first evidence at the proteomic level of massive up-regulation in a number of metabolic pathways by which the affected tissues try to compensate for the deficit in the OXPHOS machinery.
Assuntos
Anticorpos/imunologia , Regulação da Expressão Gênica , Proteínas Mitocondriais , Proteômica/métodos , Acidose Láctica/metabolismo , Animais , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Humanos , Focalização Isoelétrica , Encefalomiopatias Mitocondriais/metabolismo , Proteínas Mitocondriais/imunologia , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/química , Miocárdio/química , Fosforilação Oxidativa , CoelhosRESUMO
The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information on the protein composition of LDL may help to reveal its role in atherogenesis. Liquid-phase IEF has been used to resolve LDL proteins into well-defined fractions on the basis of pI, which improves the subsequent detection and resolution of low abundance proteins. Besides known LDL-associated proteins, this approach revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol-binding protein, and paraoxonase-1. PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. Addition of the substrate farnesylcysteine to lipoprotein resulted in a time-dependent generation of H(2)O(2) which was stronger in very low density lipoprotein (VLDL) than in LDL or HDL, reflecting the greater protein content of PCL1 in VLDL. Farnesol, a dead end inhibitor of the PCL1 reaction, reduced H(2)O(2) generation by VLDL. PCL1 is generated along with nascent lipoprotein, as shown by its presence in the lipoprotein secreted by HepG2 cells. The finding that an enzyme associated with atherogenic lipoproteins can itself generate an oxidant suggests that PCL1 may play a significant role in atherogenesis.
Assuntos
Liases de Carbono-Enxofre/metabolismo , Peróxido de Hidrogênio/metabolismo , Lipoproteínas/análise , Adulto , Aterosclerose/etiologia , Liases de Carbono-Enxofre/análise , Liases de Carbono-Enxofre/antagonistas & inibidores , Fracionamento Químico/métodos , Farneseno Álcool/metabolismo , Feminino , Humanos , Lipoproteínas/metabolismo , Lipoproteínas HDL/análise , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/análise , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/análise , Lipoproteínas VLDL/metabolismo , Masculino , Espectrometria de MassasRESUMO
Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively "tagging" them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mm, 30 min) into rat ventricular myocytes, treated them with H(2)O(2) (0.1-10,000 microm, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mm H(2)O(2) and declined sharply below basal with 10 mm treatment. Cell-wide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H(2)O(2)-induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.
Assuntos
Cicloexanonas/farmacologia , Proteômica/instrumentação , Ácidos Sulfênicos/química , Animais , Biotina/química , Cromatografia Líquida , Cicloexanonas/química , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Modelos Químicos , Células Musculares/metabolismo , Oxirredução , Estresse Oxidativo , Oxigênio/metabolismo , Proteínas/química , Proteômica/métodos , Ratos , Tripsina/químicaRESUMO
We investigated the composition of the endogenous ligand-bound type I interleukin-1 (IL-1) receptor (IL-1RI) signaling complex using immunoprecipitation and tandem mass spectrometry. Three proteins with approximate molecular masses of 60 (p60), 36 (p36), and 90 kDa (p90) became phosphorylated after treatment with IL-1. Phosphorylation in vitro of p60 has been reported previously, but its identity was unknown. We showed using tandem mass spectrometry that p60 is identical to interleukin-1 receptor-associated kinase (IRAK)-4. MS also enabled detection of IL-1, IL-1RI, IL-1 receptor accessory protein (IL-1RAcP), and myeloid differentiation primary response protein 88 (MyD88) in the complex. The p60 protein (IRAK-4) was the earliest component of the complex to be phosphorylated. Phosphorylated IRAK-4 from the receptor complex migrated more slowly in SDS-PAGE than its unphosphorylated form as did recombinant IRAK-4 autophosphorylated in vitro. Phosphorylation was restricted to serine and threonine residues. IRAK-4, p36, IL-1RAcP, and MyD88 bound to the liganded receptor within 15 s of activation by IL-1 and remained associated upon prolonged activation, suggesting that the signaling complex is very stable. The p90 phosphoprotein was only transiently associated with the receptor. This behavior and its size were consistent with it being IRAK-1. Our work revealed that liganding of IL-1RI causes its strong and stable association with IL-1RAcP, MyD88, and the previously unidentified protein p60 (IRAK-4). The only component of the IL-1RI signaling complex that dissociated is IRAK-1. Our study is therefore the first detailed description of the endogenous IL-1RI complex.
Assuntos
Proteína Acessória do Receptor de Interleucina-1/fisiologia , Quinases Associadas a Receptores de Interleucina-1/fisiologia , Espectrometria de Massas/métodos , Fator 88 de Diferenciação Mieloide/fisiologia , Receptores de Interleucina-1/metabolismo , Diferenciação Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Proteína Acessória do Receptor de Interleucina-1/química , Quinases Associadas a Receptores de Interleucina-1/química , Ligantes , Fator 88 de Diferenciação Mieloide/química , Fosfoaminoácidos/química , Fosforilação , Proteômica/métodos , Transdução de Sinais , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Convulsive status epilepticus is associated with subsequent hippocampal damage and development of mesial temporal sclerosis in a subset of individuals. The lithium pilocarpine model of status epilepticus (SE) in the rat provides a model in which to investigate the molecular and pathogenic process leading to hippocampal damage. In this study, a 2-DE-based approach was used to detect proteome changes in the hippocampus, at an early stage (2 days) after SE, when increased T2 values were detectable by magnetic resonance imaging. Gel image analysis was followed by LC-MS/MS identification of protein species that differed in abundance between pilocarpine-treated and control rats. The most significantly up-regulated species in the experimental animals was identified as heat shock 27-kDa protein, in line with findings in humans and in other experimental models of epilepsy. Additional up-regulated species included dihydropyrimidinase-related protein-2, cytoskeletal proteins (alpha-tubulin and ezrin) and dihydropteridine reductase. In summary, the hippocampus of rats subject to pilocarpine-induced SE exhibits specific changes in protein abundance, which likely relate to pathogenic, neuroprotective and neurogenic responses.
Assuntos
Hipocampo , Lítio/toxicidade , Agonistas Muscarínicos/toxicidade , Pilocarpina/toxicidade , Proteoma/análise , Estado Epiléptico , Animais , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Hipocampo/anormalidades , Hipocampo/química , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Espectrometria de Massas , Ratos , Ratos Sprague-Dawley , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismo , Estado Epiléptico/patologiaRESUMO
Here, we present the first study of a human neuromuscular disorder at transcriptional and proteomic level. Autosomal dominant facio-scapulo-humeral muscular dystrophy (FSHD) is caused by a deletion of an integral number of 3.3-kb KpnI repeats inside the telomeric region D4Z4 at the 4q35 locus. We combined a muscle-specific cDNA microarray platform with a proteomic investigation to analyse muscle biopsies of patients carrying a variable number of KpnI repeats. Unsupervised cluster analysis divides patients into three classes, according to their KpnI repeat number. Expression data reveal a transition from fast-glycolytic to slow-oxidative phenotype in FSHD muscle, which is accompanied by a deficit of proteins involved in response to oxidative stress. Besides, FSHD individuals show a disruption in the MyoD-dependent gene network suggesting a coregulation at transcriptional level during myogenesis. We also discuss the hypothesis that D4Z4 contraction may affect in trans the expression of a set of genes involved in myogenesis, as well as in the regeneration pathway of satellite cells in adult tissue. Muscular wasting could result from the inability of satellite cells to successfully differentiate into mature fibres and from the accumulation of structural damages caused by a reactive oxygen species (ROS) imbalance induced by an increased oxidative metabolism in fibres.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Lenta/patologia , Distrofia Muscular Facioescapuloumeral/metabolismo , Proteína MyoD/fisiologia , Proteínas/metabolismo , Transcrição Gênica/fisiologia , Adolescente , Adulto , Idoso , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Criança , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/patologia , Proteína MyoD/genética , Proteínas/genéticaRESUMO
The aim of the present study was to assess age-dependent changes of proteins in the vastus lateralis muscle of physically active elderly and young subjects by a combination of two-dimensional difference gel electrophoresis, SDS-PAGE and ESI-MS/MS. The differences observed in the elderly group included down-regulation of regulatory myosin light chains, particularly the phosphorylated isoforms, a higher proportion of myosin heavy chain isoforms 1 and 2A, and enhanced oxidative and reduced glycolytic capacity.
Assuntos
Envelhecimento/metabolismo , Músculo Esquelético/metabolismo , Proteoma/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional , Humanos , Dados de Sequência Molecular , Atividade Motora , Cadeias Pesadas de Miosina/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for alpha-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of beta-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Oxidantes/farmacologia , Animais , Células Cultivadas , Dissulfetos , Ativação Enzimática/efeitos dos fármacos , Coração , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , Masculino , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Oxirredução , Fosforilação , Subunidades Proteicas , Transporte Proteico , Ratos , Ratos Wistar , Miosinas VentricularesRESUMO
Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteoma , Streptococcus pneumoniae/química , Sequência de Aminoácidos , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Ácidos Cólicos/química , Ditiotreitol/química , Eletroforese em Gel Bidimensional , Dados de Sequência Molecular , Dodecilsulfato de Sódio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ureia/químicaRESUMO
Eukaryotic cells plasma membranes are organized into microdomains of specialized function such as lipid rafts and caveolae, with a specific lipid composition highly enriched in cholesterol and glycosphingolipids. In addition to their role in regulating signal transduction, multiple functions have been proposed, such as anchorage of receptors, trafficking of cholesterol, and regulation of permeability. However, an extensive understanding of their protein composition in human heart, both in failing and non-failing conditions, is not yet available. Membrane microdomains were isolated from left ventricular tissue of both failing (n = 15) and non-failing (n = 15) human hearts. Protein composition and differential protein expression was explored by comparing series of 2-D maps and subsequent identification by LC-MS/MS analysis. Data indicated that heart membrane microdomains are enriched in chaperones, cytoskeletal-associated proteins, enzymes and protein involved in signal transduction pathway. In addition, differential protein expression profile revealed that 30 proteins were specifically up- or down-regulated in human heart failure membrane microdomains. This study resulted in the identification of human heart membrane microdomain protein composition, which was not previously available. Moreover, it allowed the identification of multiple proteins whose expression is altered in heart failure, thus opening new perspectives to determine which role they may play in this disease.
Assuntos
Insuficiência Cardíaca/metabolismo , Microdomínios da Membrana/química , Proteínas de Membrana/análise , Proteômica/métodos , Adulto , Sequência de Aminoácidos , Estudos de Casos e Controles , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Feminino , Insuficiência Cardíaca/genética , Ventrículos do Coração/química , Humanos , Immunoblotting , Masculino , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Mapeamento de Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In this case report we studied alterations in mitochondrial proteins in a patient suffering from recurrent profound muscle weakness, associated with ethylmalonic-adipic aciduria, who had benefited from high dose of riboflavin treatment. Morphological and biochemical alterations included muscle lipid accumulation, low muscle carnitine content, reduction in fatty acid beta-oxidation and reduced activity of complexes I and II of the respiratory chain. Riboflavin therapy partially or totally reversed these symptoms and increased the level of muscle flavin adenine dinucleotide, suggesting that aberrant flavin cofactor metabolism accounted for the disease. Proteomic investigation of muscle mitochondria revealed decrease or absence of several flavoenzymes, enzymes related to flavin cofactor-dependent mitochondrial pathways and mitochondrial or mitochondria-associated calcium-binding proteins. All these deficiencies were completely rescued after riboflavin treatment. This study indicates for the first time a profound involvement of riboflavin/flavin cofactors in modulating the level of a number of functionally coordinated polypeptides involved in fatty acyl-CoA and amino acid metabolism, extending the number of enzymatic pathways altered in riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.
Assuntos
Acil-CoA Desidrogenases/deficiência , Músculo Esquelético/enzimologia , Riboflavina/uso terapêutico , Aminoácidos/metabolismo , Complexo I de Transporte de Elétrons/deficiência , Complexo II de Transporte de Elétrons/deficiência , Eletroforese em Gel Bidimensional , Ácidos Graxos/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Histocitoquímica , Humanos , Metabolismo dos Lipídeos , Masculino , Pessoa de Meia-Idade , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/metabolismo , Modelos Biológicos , Músculo Esquelético/metabolismo , Oxirredução , Proteômica , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Glutathione disulfide (GSSG) accumulates in cells under an increased oxidant load, which occurs during neurohormonal or metabolic stimulation as well as in many disease states. Elevated GSSG promotes protein S-glutathiolation, a reversible post-translational modification, which can directly alter or regulate protein function. We developed novel strategies for the study of protein S-glutathiolation that involved the simple synthesis of N,N-biotinyl glutathione disulfide (biotin-GSSG). Biotin-GSSG treatment of cells mimics a defined component of oxidative stress, namely a shift in the glutathione redox couple to the oxidized disulfide state. This induces widespread protein S-glutathiolation, which was detected on non-reducing Western blots probed with streptavidin-horseradish peroxidase and imaged using confocal fluorescence microscopy and ExtrAvidin-FITC. S-Glutathiolated proteins were purified using streptavidin-agarose and identified using proteomic methods. We conclude that biotin-GSSG is a useful tool in the investigation of protein S-glutathiolation and offers significant advantages over conventional methods or antibody-based strategies. These novel approaches may find widespread utility in the study of disease or redox signaling models where GSSG accumulation occurs.
Assuntos
Biotinilação , Dissulfeto de Glutationa/química , Proteínas/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Masculino , Dados de Sequência Molecular , Miócitos Cardíacos/metabolismo , Proteínas/análise , Proteínas/isolamento & purificação , Ratos , Ratos WistarRESUMO
Mutations in dysferlin gene cause several types of muscular dystrophy in humans, including the limb-girdle muscular dystrophy type 2B and the distal muscular dystrophy of Miyoshi. The dysferlin gene product is a membrane-associated protein belonging to the ferlins family of proteins. The function of the dysferlin protein and the cause of deterioration and regression of muscle fibres in its absence, are incompletely known. A functional clue may be the presence of six hydrophilic domains, C2, that bind calcium and mediate the interaction of proteins with cellular membranes. Dysferlin seems to be involved in the membrane fusion or repair. Molecular diagnosis of dysferlinopathies is now possible and the types of gene alterations that have been characterized so far include missense mutations, deletions and insertions.
Assuntos
Proteínas de Membrana/genética , Proteínas Musculares/genética , Distrofias Musculares/fisiopatologia , Proteoma , Sequência de Aminoácidos , Disferlina , Eletroforese em Gel Bidimensional , Humanos , Dados de Sequência Molecular , Distrofias Musculares/genética , MutaçãoRESUMO
Functional characterization of muscle fibers relies on ATPase activity and on differential measurements of metabolic proteins, including mitochondrial and glycolytic enzymes, glucose, lactate and lactic acid transporters, calcium cycling proteins and components of the contractile machinery. The recent introduction of microarray technology has enabled detailed gene expression studies under different physiological and pathological conditions, thus generating novel hypotheses on muscle function. However, microarray approaches are limited by the incomplete genome coverage of currently available chips, and by poor correlation between mRNA concentration and protein expression level. We have used 2-DE and MS to build a reference map of proteins from rat mixed gastrocnemius and soleus muscle, and to assess qualitative and quantitative differences in protein distribution between these two functionally dissimilar muscles. More than 800 spots on each gel were detected by silver staining, of which 167 were excised, digested in-gel with trypsin and analyzed by ESI-MS/MS. One hundred and twenty eight distinct gene products were identified, including metabolic, transport and contractile proteins. Forty one spots displayed differences in relative expression level between mixed gastrocnemius and soleus samples. These data not only enable differentiation of functionally distinct slow-twitch and fast-twitch fiber types, but also provide tools for investigating muscle plasticity in response to physiological and environmental conditions such as aging or hypoxia.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas Musculares/química , Músculo Esquelético/química , Músculo Esquelético/fisiologia , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Cadeias Leves de Miosina/metabolismo , Oxigênio/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Microparticles (MP) are small membrane vesicles that are released from cells upon activation or during apoptosis. Cellular MP in body fluids constitute a heterogeneous population, differing in cellular origin, numbers, size, antigenic composition and functional properties. MP support coagulation by exposure of tissue factor (TF), the initiator of coagulation in vivo. Moreover, MP may transfer bioactive molecules to other cells, thereby stimulating them to produce cytokines, cell-adhesion molecules, growth factors and TF, and modulate endothelial functions. However, a comprehensive characterization of the antigenic composition of MP has been poorly defined. This study describes the protein composition of endothelial cell (EC)-derived MP (EMP) using a proteomic approach. MS analysis indicated the presence of newly described protein such as metabolic enzymes, proteins involved in adhesion and fusion processes, members of protein folding event, cytoskeleton associated proteins and nucleosome. In conclusion, circulating EMP behave as an actual storage pool, able to disseminate blood-borne TF activity and other bioactive effectors, as confirmed by our experiments showing an increased procoagulant activity of EC exposed to EMP.
Assuntos
Coagulação Sanguínea/fisiologia , Membrana Celular/ultraestrutura , Endotélio Vascular/fisiologia , Tromboplastina/análise , Sequência de Aminoácidos , Apoptose , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/ultraestrutura , Enzimas/química , Enzimas/isolamento & purificação , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Proteínas/química , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fator de Necrose Tumoral alfa/farmacologia , Veias UmbilicaisRESUMO
We present reference maps of the mouse serum proteome (run under reducing and non-reducing conditions), from control animals, from mice injected with lipopolysaccharide (LPS) to induce systemic inflammation, and from mice transgenic for human apolipoproteins A-I and A-II. Seventy-seven spots/spot chains from the reducing gels were identified by HPLC MS/MS, representing 28 distinct proteins, including a species-specific protease inhibitor, contrapsin, and high levels of carboxylesterase. The concentrations of acute-phase reactants were monitored for 96 h after LPS challenge. The greatest changes (four-fold 48 h after LPS administration) were observed for haptoglobin and hemopexin. Orosomucoid/alpha(1)-acid glycoprotein and apolipoprotein A-I increased steadily, to 50-60% above baseline at 96 h from stimulation. In mice transgenic for human apolipoprotein A-I the levels of expression of orosomucoid/alpha(1)-acid glycoprotein, alpha(1)-macroglobulin, esterase, kininogen and contrapsin were altered compared to knockout mice lacking apolipoprotein A-I. In contrast, except for the presence of apolipoprotein A-II, no statistically significant difference was observed in mice transgenic for human apolipoprotein A-II.
Assuntos
Proteínas de Fase Aguda/metabolismo , Reação de Fase Aguda , Apolipoproteína A-II/genética , Apolipoproteína A-I/genética , Lipopolissacarídeos , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Haptoglobinas/metabolismo , Hemopexina/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Orosomucoide/metabolismo , Valores de ReferênciaRESUMO
In an accompanying study (in this issue, DOI 10.1002/pmic.200402044), we have characterised the proteome of Sca-1(+) progenitor cells, which may function as precursors of vascular smooth muscle cells (SMCs). In the present study, we have analysed and mapped protein expression in aortic SMCs of mice, using 2-DE, MALDI-TOF MS and MS/MS. The 2-D system comprised a non-linear immobilised pH 3-10 gradient in the first dimension (separating proteins with pI values of pH 3-10), and 12%T SDS-PAGE in the second dimension (separating proteins in the range 15,000-150,000 Da). Of the 2400 spots visualised, a subset of 267 protein spots was analysed, with 235 protein spots being identified corresponding to 154 unique proteins. The data presented here are the first map of aortic SMCs and the most extensive analysis of SMC proteins published so far. This valuable tool should provide a basis for comparative studies of protein expression in vascular smooth muscle of transgenic mice and is available on our website hhtp://www.vascular-proteomics.com.