RESUMO
Recent studies support the role of cysteine oxidation in actin cytoskeleton reorganization during cell adhesion. The aim of this study was to explain whether protein disulfide isomerase (PDI) is responsible for the thiol-disulfide rearrangement in the ß-actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with ß-actin with a molecular mass of 110 kDa. Specific interaction of both proteins was demonstrated by a solid phase binding assay, surface plasmon resonance analysis, and immunoprecipitation experiments. Second, using confocal microscopy, we found that both proteins colocalized when spreading MEG-01 cells on fibronectin. Colocalization of PDI and ß-actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-αIIbß3 antibodies. Consequently, down-regulation of PDI expression by antisense oligonucleotides impaired the spreading of cells and initiated reorganization of the cytoskeleton. Third, because of transfection experiments followed by immunoprecipitation and confocal analysis, we provided evidence that PDI binds to the ß-actin Cys(374) thiol. Formation of the ß-actin-PDI complex was mediated by integrin-dependent signaling in response to the adhesion of cells to the extracellular matrix. Our data suggest that PDI is released from subcellular compartments to the cytosol and translocated toward the periphery of the cell, where it forms a disulfide bond with ß-actin when MEG-01 cells adhere via the αIIbß3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in ß-actin via a redox-dependent mechanism.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Transdução de Sinais/fisiologia , Actinas/genética , Antineoplásicos/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular , Citoesqueleto/genética , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Oligopeptídeos/farmacologia , Oxirredução/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
UNLABELLED: The aim of the study is proteomic analysis of the plasma profile in children with recurrent bone fractures. The study involved 16 children: 6 patients with recurrent low-energy fractures and normal bone mass and 10 with osteogenesis imperfecta. In the analysis of the protein profile, the two-dimensional protein electrophoresis was used (Ettan DALT II, Amersham Bioscience). The images of protein gels were compared with controls. The protein spots with changed expression were cut from the gel and the amino acid sequence was analyzed with the mass spectrometry method (Q-Tof Premier(TM) API MASS SPECTROMETR, Waters) for protein identification. The most prevalent protein with changed expression, with respect to controls, was haptoglobin observed in 6 patients with a severe form of osteogenesis imperfecta. Increased haptoglobin concentration in these patients was confirmed by the ELISA method. Peptides corresponding to alpha-1 acid glycoprotein and serum amyloid P-component, apolipoprotein A-I, and transthyretin were detected in one, two and three children, respectively. CONCLUSIONS: 1) The results show increased haptoglobin which may be suggestive of an inflammatory component taking part in the course of osteogenesis imperfecta. 2) Further studies to explain the possible relationship of this protein with increased bone fragility are necessary.
Assuntos
Fraturas Ósseas/sangue , Haptoglobinas/metabolismo , Osteogênese Imperfeita/patologia , Proteoma/análise , Adolescente , Sequência de Aminoácidos , Apolipoproteína A-I/metabolismo , Densidade Óssea , Estudos de Casos e Controles , Criança , Pré-Escolar , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Fraturas Ósseas/metabolismo , Fraturas Ósseas/patologia , Humanos , Lactente , Masculino , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Osteogênese Imperfeita/sangue , Osteogênese Imperfeita/metabolismo , Mapeamento de Peptídeos/métodos , Pré-Albumina/metabolismo , Componente Amiloide P Sérico/metabolismoRESUMO
Recent evidence supports a role of protein-disulfide isomerase (PDI) in redox-controlled remodeling of the exofacial domains of α(IIb)ß(3) in blood platelets. The aim of this study was to explain whether Ero1α can be responsible for extracellular reoxidation of the PDI active site. We showed that Ero1α can be found on platelets and is rapidly recruited to the cell surface in response to platelet agonists. It is physically associated with PDI and α(IIb)ß(3), as suggested by colocalization analysis in confocal microscopy and confirmed by immunoprecipitation experiments. Apart from monomeric oxidized Ero1α, anti-α(IIb)ß(3) immunoprecipitates showed the presence of several Ero1α-positive bands that corresponded to the complexes α(IIb)ß(3)-PDI-Ero1α, PDI-Ero1α, and Ero1α-Ero1α dimers. It binds more efficiently to the activated α(IIb)ß(3) conformer, and its interaction is inhibited by RGD peptides. Ero1α appears to be involved in the regulation of α(IIb)ß(3) receptor activity because of the following: (a) blocking the cell surface Ero1α by antibodies leads to a decrease in platelet aggregation in response to agonists and a decrease in fibrinogen and PAC-1 binding, and (b) transfection of MEG01 with Ero1α increases α(IIb)ß(3) receptor activity, as indicated by increased binding of fibrinogen.
Assuntos
Plaquetas/metabolismo , Glicoproteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Oxirredutases/metabolismo , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Domínio Catalítico , Fibrinogênio/genética , Fibrinogênio/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Complexos Multiproteicos/genética , Oligopeptídeos/farmacologia , Oxirredução/efeitos dos fármacos , Oxirredutases/genética , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Isomerases de Dissulfetos de Proteínas/genéticaRESUMO
A novel affinity purification method for rapid isolation of vitronectin (VN) from human plasma is described. Recently we have used phage display technology to obtain clones expressing peptides with high binding activity for VN. The isolated "strong VN binders" were covalently coupled to CNBr-activated Sepharose. Human plasma was applied to the column and bound VN was eluted using 0.5 M acetic acid, giving purity exceeding 90%. The developed method is a convenient alternative to conventional antibody-antigen affinity chromatography techniques for purification of VN, as it offers low ligand cost, is rapid and ensures good protein recovery from human plasma.
