RESUMO
It has been postulated that post-translational modifications and relocalization of proteins during apoptosis may lead to presentation of these molecules to the immune system in such a way that normal mechanisms of tolerance are bypassed. In the present study, Jurkat cells were induced to undergo apoptosis by treatment with the chemotherapeutic agent Ara-C. BALB/c mice were then immunized with the apoptotic cells and hybridomas were generated. Using an indirect immunofluorescence assay, the monoclonal antibodies produced were screened by flow cytometry for those monoclonal antibodies demonstrating reactivity with permeabilized apoptotic Jurkat cells but not with non-permeabilized normal or apoptotic Jurkat cells. Of 281 monoclonal antibodies, 20 monoclonal antibodies with these properties were selected for further analysis. Using 32P- or 35S-metabolically labelled Jurkat cells, these selected monoclonal antibodies were screened for their ability to recognize autoantigens by immunoprecipitation and Western blotting. Well characterized autoimmune sera were then used to confirm the identity of autoantigens by immunoblotting. We demonstrate that immunization of normal mice with apoptotic Jurkat cells results in the formation of antibodies targeting multiple autoantigens or autoantigen complexes, including Ku, rRNPs, snRNPs and vimentin. These findings are consistent with the hypothesis that apoptosis can contribute to the development of autoimmunity.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Nucleares , Apoptose , Autoanticorpos/imunologia , Autoantígenos/imunologia , DNA Helicases , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Autoanticorpos/biossíntese , Proteínas de Ligação a DNA/imunologia , Humanos , Imunização , Immunoblotting/métodos , Marcação por Isótopo , Células Jurkat , Autoantígeno Ku , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Nucleares/imunologia , Testes de Precipitina/métodos , Ribonucleoproteína Nuclear Pequena U1/imunologia , Ribonucleoproteína Nuclear Pequena U2/imunologia , Proteínas Ribossômicas/imunologia , Radioisótopos de Enxofre , Vimentina/imunologiaRESUMO
Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.
Assuntos
Proteínas de Ciclo Celular , Proteínas Culina , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Helminto/metabolismo , Proteínas I-kappa B , Peptídeo Sintases/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Helminto/genética , Humanos , Cinética , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Proteína NEDD8 , Fosforilação , Proteínas Ligases SKP Culina F-Box , Alinhamento de Sequência , Transfecção , beta Catenina , Proteínas Contendo Repetições de beta-TransducinaRESUMO
Proteins cleaved by apoptotic caspases are commonly recognized by autoantibodies found in the serum of patients with rheumatic disease. We report that the 72-kDa signal recognition particle (SRP) protein, a rare target of autoantibodies found in the serum of patients with dermatomyositis and systemic lupus erythematosus, is rapidly cleaved in Jurkat T cells treated with apoptotic (i.e. Fas ligation, treatment with gamma or ultraviolet radiation, or co-culture with anisomycin or staurosporine) but not proliferative (CD3 cross-linking) stimuli. Cleavage of SRP 72 produces a 66-kDa amino-terminal fragment and a 6-kDa carboxyl-terminal fragment that is selectively phosphorylated on serine residues. Cleavage of SRP 72 is prevented by chemical and peptide caspase inhibitors, and by overexpression of bcl-2, an inhibitor of apoptotic cell death. Analysis of the carboxyl terminus of SRP 72 has identified a putative cleavage site (SELD/A) for group III caspases, and carboxyl-terminal serine residues that are highly conserved in phylogeny. Both serine phosphorylation and caspase cleavage of SRP 72 are observed in cells derived from human, dog, rat, and mouse. Canine SRP 72 is cleaved in vitro by recombinant caspase 3 but retains the ability to mediate transport of a signal peptide-containing protein into the endoplasmic reticulum lumen. The 72-kDa component of the SRP joins a growing list of autoantigens that undergo post-translational modifications during programmed cell death.
Assuntos
Apoptose , Autoantígenos/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Linfócitos T/imunologia , Sequência de Aminoácidos , Autoanticorpos , Inibidores de Caspase , Dermatomiosite/imunologia , Raios gama/efeitos adversos , Humanos , Células Jurkat/imunologia , Células Jurkat/efeitos da radiação , Lúpus Eritematoso Sistêmico/imunologia , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Especificidade da Espécie , Linfócitos T/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Receptor fas/metabolismoRESUMO
Proteins subject to proteolysis or phosphorylation during apoptosis are commonly precipitated by autoantibodies found in the serum of patients with systemic lupus erythematosus (SLE). We screened a panel of murine monoclonal and human monospecific sera reactive with known autoantigens for their ability to selectively precipitate phosphoproteins from apoptotic Jurkat T cell lysates. Sera known to recognize the U1-small nuclear ribonucleoprotein (snRNP) complex (confirmed by their ability to precipitate U1-snRNA) selectively precipitated a phosphoprotein complex (pp54, pp42, pp34, and pp23) from apoptotic lysates. Monoclonal antibodies reactive with U1-snRNP proteins precipitated the same phosphoprotein complex from apoptotic lysates. The phosphorylation and/or recruitment of these proteins to the U1-snRNP complex is induced by multiple apoptotic stimuli (e.g., Fas ligation, gamma irradiation, or UV irradiation), and is blocked by overexpression of bcl-2. The U1-snRNP-associated phosphoprotein complex is immunoprecipitated by monoclonal antibodies reactive with serine/arginine (SR) proteins that comprise a structurally related family of splicing factors. The association of phosphorylated SR proteins with the U1-snRNP complex in cells undergoing apoptosis suggests a mechanism for regulation of alternative splicing of apoptotic effector molecules.
Assuntos
Reações Antígeno-Anticorpo/imunologia , Apoptose/imunologia , Autoantígenos/imunologia , Proteínas Nucleares/imunologia , Fosfoproteínas/imunologia , Proteínas de Ligação a RNA/imunologia , Ribonucleoproteína Nuclear Pequena U1/imunologia , Ribonucleoproteínas , Animais , Anticorpos Monoclonais/imunologia , Humanos , Soros Imunes/imunologia , Células Jurkat , Substâncias Macromoleculares , Camundongos , Splicing de RNA/imunologia , RNA Nuclear Pequeno/imunologia , Fatores de Processamento de Serina-Arginina , Spliceossomos/imunologia , Receptor fas/imunologiaRESUMO
Proteins cleaved by interleukin-1 beta converting enzyme family proteases during apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus (SLE). We have tested the possibility that proteins phosphorylated in cells undergoing apoptosis are also targets for autoantibody production in patients with autoimmune disease. Sera from 9/12 patients containing antinuclear antibodies (10/12 meeting diagnostic criteria for SLE or a lupus overlap syndrome), precipitated new phosphoproteins from lysates derived from Jurkat T cells treated with apoptotic stimuli (i.e., Fas-ligation, gamma irradiation, ultraviolet irradiation), but not with an activation (i.e., CD3-ligation) stimulus. Sera derived from individual patients precipitated different combinations of seven distinct serine-phosphorylated proteins. None of these phosphoproteins were included in precipitates prepared using sera from patients with diseases that are not associated with autoantibody production or using serum from rheumatoid arthritis patients. Protein phosphorylation precedes, or is coincident with, the induction of DNA fragmentation, and is not observed when apoptosis is inhibited by overexpression of bcl-2. Serum from four patients precipitated a serine/threonine kinase from apoptotic cell lysates that phosphorylates proteins of 23-, 34-, and 46-kD in in vitro kinase assays. Our results suggest that proteins phosphorylated during apoptosis may be preferred targets for autoantibody production in patients with SLE.