RESUMO
The N-end rule states that the half-life of a protein is determined by the nature of its amino-terminal residue. Eukaryotes and prokaryotes use N-terminal destabilizing residues as a signal to target proteins for degradation by the N-end rule pathway. In eukaryotes an E3 ligase, N-recognin, recognizes N-end rule substrates and mediates their ubiquitination and degradation by the proteasome. In Escherichia coli, N-end rule substrates are degraded by the AAA + chaperone ClpA in complex with the ClpP peptidase (ClpAP). Little is known of the molecular mechanism by which N-end rule substrates are initially selected for proteolysis. Here we report that the ClpAP-specific adaptor, ClpS, is essential for degradation of N-end rule substrates by ClpAP in bacteria. ClpS binds directly to N-terminal destabilizing residues through its substrate-binding site distal to the ClpS-ClpA interface, and targets these substrates to ClpAP for degradation. Degradation by the N-end rule pathway is more complex than anticipated and several other features are involved, including a net positive charge near the N terminus and an unstructured region between the N-terminal signal and the folded protein substrate. Through interaction with this signal, ClpS converts the ClpAP machine into a protease with exquisitely defined specificity, ideally suited to regulatory proteolysis.
Assuntos
Proteínas de Transporte/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Escherichia coli/genética , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: The chemokine receptor CXCR4 (a receptor for the Cys-X-Cys class of chemokines) is a CD4-associated coreceptor for T-cell-tropic strains of human immunodeficiency virus 1 (HIV-1) and represents a target for antiviral therapy. Infection by T-tropic HIV-1 can be blocked by stromal-cell-derived factor-1 (SDF-1), the natural ligand of CXCR4. The broad variety of cells expressing CXCR4 and the perturbations observed in mice deficient for SDF-1 suggest that antiviral compounds antagonizing the signalling activity of CXCR4 might have severe side effects in vivo. Compounds that interfere selectively with HIV entry and not with SDF-1 signalling would therefore be useful. RESULTS: A series of peptides, each of 13 residues, spanning the whole SDF-1alpha sequence were tested for their ability to block HIV-1 infection. The antiviral and signalling properties of SDF-1 were retained by a peptide corresponding to its amino terminus. Removal of the first two residues resulted in an antiviral antagonist of the SDF-1-CXCR4 signalling pathway. We prepared 234 single-substitution analogues and identified one antiviral analogue that had drastically reduced agonistic or antagonistic properties. The antiviral peptides competed with the monoclonal antibody 12G5 for CXCR4 binding. Their antiviral activity seems to be due to receptor occupancy rather than induction of receptor endocytosis. CONCLUSIONS: The amino terminus of the SDF-1 chemokine is sufficient for signal transduction via CXCR4 and for inhibition of HIV-1 entry, but these activities could be dissociated in a peptide analogue. This peptide represents a lead molecule for the design of low molecular weight antiviral drugs.
Assuntos
Fármacos Anti-HIV/farmacologia , Quimiocinas CXC/farmacologia , HIV-1/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Fármacos Anti-HIV/química , Quimiocina CXCL12 , Quimiocinas CXC/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Infecções por HIV/prevenção & controle , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Receptores CXCR4/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The SmD1 protein is a specific target for the autoantibody response in SLE. To further analyze this reactivity epitope, mapping was performed with cellulose-bound 13-mer peptides overlapping 10 amino acids (aa). In this initial approach, 4 out of 15 SLE sera recognized more than five overlapping peptides of the SmD1 C-terminus. Therefore, longer oligopeptides of up to 37 aa of this region were generated and probed for as antigens by ELISA. For the SmD1 aa 83-119 polypeptide, there was a striking increase of reactivity with 70.0% positive reactions out of 167 SLE sera. In contrast, 105 healthy control sera were negative, and only 8.3% of sera from patients with other inflammatory diseases (n = 267) exhibited a response, which was of low level only. The anti-SmD183-119 reactivity was significantly higher in anti-dsDNA antibody positive vs. negative sera (P < 0.001) and correlated with disease activity. Four of five human monoclonal anti-dsDNA antibodies also reacted with SmD183-119. The specificity for SmD1 was demonstrated by inhibition experiments and immunization of rabbits with SmD183-119 inducing SmD1-specific antibodies. In conclusion, the SmD183-119 peptide was demonstrated to be an important and highly specific target of the autoimmune response in SLE. The high sensitivity of this ELISA probably depends on a conformational epitope, which appears not to be accessible in the full-size SmD1 protein.
Assuntos
Especificidade de Anticorpos , Autoanticorpos/sangue , Autoantígenos/imunologia , Epitopos , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/imunologia , Ribonucleoproteínas Nucleares Pequenas , Sequência de Aminoácidos , Autoantígenos/química , Autoimunidade , DNA/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/imunologia , Fragmentos de Peptídeos/química , Conformação Proteica , Sensibilidade e Especificidade , Proteínas Centrais de snRNPRESUMO
Signal transducer and transcription (STAT) factors are activated by tyrosine phosphorylation in response to a variety of cytokines, growth factors, and hormones. Tyrosine phosphorylation triggers dimerization and nuclear translocation of these transcription factors. In this study, the functional role of carboxy-terminal portions of the STAT family member acute-phase response factor/Stat3 in activation, dimerization, and transactivating potential was analyzed. We demonstrate that truncation of 55 carboxy-terminal amino acids causes constitutive activation of Stat3 in COS-7 cells, as is known for the Stat3 isoform Stat3beta. By the use of deletion and point mutants, it is shown that both carboxy- and amino-terminal portions of Stat3 are involved in this phenomenon. Dimerization of Stat3 was blocked by point mutations affecting residues both in the vicinity of the tyrosine phosphorylation site (Y705) and more distant from this site, suggesting that multiple interactions are involved in dimer formation. Furthermore, by reporter gene assays we demonstrate that carboxy-terminally truncated Stat3 proteins are incapable of transactivating an interleukin-6-responsive promoter in COS-7 cells. In HepG2 hepatoma cells, however, these truncated Stat3 forms transmit signals from the interleukin-6 signal transducer gp130 equally well as does full-length Stat3. We conclude that, dependent on the cell type, different mechanisms allow Stat3 to regulate target gene transcription either with or without involvement of its putative carboxy-terminal transactivation domain.
Assuntos
Proteínas de Fase Aguda/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transativadores/metabolismo , Ativação Transcricional/fisiologia , Proteínas de Fase Aguda/genética , Sequência de Aminoácidos , Animais , Células COS , Carcinoma Hepatocelular , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Dimerização , Humanos , Interleucina-6/metabolismo , Neoplasias Hepáticas , Dados de Sequência Molecular , Fosforilação , Mutação Puntual , Regiões Promotoras Genéticas/genética , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Deleção de Sequência , Transativadores/genética , Células Tumorais Cultivadas , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
We synthetically reconstructed a discontinuous binding site on interleukin-10 (IL-10) that recognizes the neutralizing anti-IL-10 antibody CB/RS/1. To design the 32-mer IL-10 mimic, a discontinuous interaction site on IL-10 was mapped, and binding studies with epitope-derived peptides led to specific replacement of several amino acids. Both parts of the interaction site were combined by addition of a linker molecule. Systematic analoging of the combined molecule then led to introduction of several additional substitutions in both regions and the linker. All possible disulfide bridge-containing variants of the 32-mer were tested by binding studies. Parallel syntheses were performed on continuous cellulose membranes by spot synthesis. As a result, a conformationally stabilized IL-10-derived molecule was obtained that both binds to and neutralizes the biological activity of CB/RS/1 in the low nanomolar range. This synthetic approach is a powerful alternative to phage display methods for the design of protein mimics.
Assuntos
Sítios de Ligação/fisiologia , Interleucina-10/química , Substituição de Aminoácidos , Ligação Competitiva , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Epitopos , Humanos , Cinética , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/metabolismo , Biossíntese Peptídica , Mapeamento de Peptídeos , Fator de Necrose Tumoral alfa/metabolismoAssuntos
Mapeamento de Epitopos/métodos , Citometria de Fluxo/métodos , Antígeno HLA-A2/química , Antígeno HLA-B7/química , Linfócitos T/imunologia , Sequência de Aminoácidos , Epitopos/química , Humanos , Complexo Principal de Histocompatibilidade , Dados de Sequência Molecular , Biblioteca de Peptídeos , Linfócitos T Citotóxicos/imunologiaRESUMO
BACKGROUND: The tremendous increase in sequential and structural information is a challenge for computer-assisted modelling to predict the binding modes of interacting biomolecules. One important area is the structural understanding of protein-peptide interactions, information that is increasingly important for the design of biologically active compounds. RESULTS: We predicted the three-dimensional structure of a complex between the monoclonal antibody TE33 and its cholera-toxin-derived peptide epitope VPGSQHID. Using the internal coordinate mechanics (ICM) method of flexible docking, the bound conformation of the initially extended peptide epitope to the antibody crystal or modelled structure reproduced the known binding conformation to a root mean square deviation of between 1.9 A and 3.1 A. The predicted complexes are in good agreement with binding data obtained from substitutional analyses in which each epitope residue is replaced by all other amino acids. Furthermore, a de novo prediction of the recently discovered TE33-binding D peptide dwGsqhydp (single-letter amino acid code where D amino acids are represented by lower-case letters) explains results obtained from binding studies with 172 peptide analogues. CONCLUSIONS: Despite the difficulties arising from the huge conformational space of a peptide, this approach allowed the prediction of the correct binding orientation and the majority of essential binding features of a peptide-antibody complex.
Assuntos
Anticorpos Monoclonais/imunologia , Toxina da Cólera/imunologia , Fragmentos de Peptídeos/imunologia , Simulação por Computador , Cristalografia por Raios X , Epitopos/imunologia , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
This study examined the impact of the tyrosine kinase Lyn on erythropoietin-induced intracellular signaling in erythroid cells. In J2E erythroleukemic cells, Lyn coimmunoprecipitated with numerous proteins, including SHP-1, SHP-2, ras-GTPase-activating protein, signal transducers and activators of transcription (STAT) 5a, STAT5b, and mitogen-activated protein kinase; however, introduction of a dominant-negative Lyn (Y397F Lyn) inhibited the interaction of Lyn with all of these molecules except SHP-1. Cells containing the dominant-negative Lyn displayed altered intracellular phosphorylation patterns, including mitogen-actiated protein kinase, but not erythropoietin receptor, Janus-activated kinase (JAK) 2, or STAT5. As a consequence, erythropoietin-initiated differentiation and basal proliferation were severely impaired. Y397F Lyn reduced the protein levels of erythroid transcription factors erythroid Kruppel-like factor and GATA-1 up to 90%, which accounts for the inability of J2E cells expressing Y397F Lyn to synthesize hemoglobin. Although Lyn was shown to bind several sites on the cytoplasmic domain of the erythropoietin receptor, it was not activated when a receptor mutated at the JAK2 binding site was ectopically expressed in J2E cells indicating that JAK2 is the primary kinase in erythropoietin signaling and that Lyn is a secondary kinase. In normal erythroid progenitors, erythropoietin enhanced phosphorylation of Lyn; moreover, exogenous Lyn increased colony forming unit-erythroid, but not burst forming uniterythroid, colonies from normal progenitors, demonstrating a stage-specific effect of the kinase. Significantly, altering Lyn activity in J2E cells had a profound effect on the development of erythroleukemias in vivo: the mortality rate was markedly reduced and latent period extended when either wild-type Lyn or Y397F Lyn was introduced into these cells. Taken together, these data show that Lyn plays an important role in intracellular signaling in nontransformed and leukemic erythroid cells.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/enzimologia , Leucemia Eritroblástica Aguda/enzimologia , Proteínas Proto-Oncogênicas , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Ativação Enzimática , Eritropoetina/farmacologia , Hemoglobinas/biossíntese , Janus Quinase 2 , Leucemia Eritroblástica Aguda/patologia , Fígado/citologia , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptores da Eritropoetina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Hsp70 chaperones assist protein folding by reversible interaction with extended hydrophobic segments of substrate polypeptides. We investigated the contribution of three structural elements of the substrate- binding cavity of the Escherichia coli homologue, DnaK, to substrate specificity by investigating mutant DnaK proteins for binding to cellulose-bound peptides. Deletion of the C-terminal subdomain (Delta539-638) and blockage of the access to the hydrophobic pocket in the substrate-binding cavity (V436F) did not change the specificity, although the latter exchange reduced the affinity to all peptides investigated. Mutations (A429W, M404A/A429W) that affect the formation of a hydrophobic arch spanning over the bound substrate disfavored DnaK binding, especially to peptides with short stretches of consecutive hydrophobic residues flanked by acidic residues, while binding to most other peptides remained unchanged. The arch thus contributes to the substrate specificity of DnaK. This finding is of particular interest, since of all the residues of the substrate-binding cavity that contact bound substrate, only the arch-forming residues show significant variation within the Hsp70 family.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sítios de Ligação , Escherichia coli/genética , Proteínas de Choque Térmico HSP70/genética , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato/genéticaRESUMO
The mitochondrial import receptor of 70 kDa, Tom70, preferentially recognizes precursors of membrane proteins with internal targeting signals. We report the identification of a stably folded 25 kDa core domain located in the middle portion of Tom70 that contains two of the seven tetratricopeptide repeat motifs of the receptor. The core domain binds non-cleavable and cleavable preproteins carrying internal targeting signals with a specificity indistinguishable from the full-length receptor. Competition studies indicate that both types of preproteins interact with overlapping binding sites of the core domain and that at least one additional interaction site is present in the full-length receptor. We suggest a model of Tom70 function in import of membrane proteins whereby a hydrophobic preprotein concomitantly interacts with several binding sites of the receptor.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ligação Competitiva/efeitos dos fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Dobramento de Proteína , Precursores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Deleção de Sequência/genética , Especificidade por Substrato , Termodinâmica , Tripsina/metabolismoRESUMO
The E. coli heat shock response is regulated at the transcriptional level through stress-dependent controls of the heat shock promoter-specific sigma32 subunit of RNA polymerase. A key aspect of this regulation, the sensing of stress and transmission of this information to sigma32, involves the chaperone system formed by the DnaK, DnaJ and GrpE heat shock proteins. This system mediates stress- dependent controls of levels and activity of sigma32 which rely, at least in part, on direct association of DnaK and DnaJ with sigma32. We identified DnaK binding sites within the sigma32 sequence by probing a cellulose-bound peptide library scanning sigma32. Two sites with high affinity for DnaK, containing the motifs RKLFFNLR and LRNWRIVK, were located centrally and peripherally, respectively, to the region C of sigma32, previously implicated genetically in chaperone-dependent control of sigma32 levels. Cloning and sequencing of rpoH homologs from five Gram-negative proteobacteria revealed that region C, including the DnaK binding motif central to it, is highly conserved among sigma32 homologs but missing in the other sigma factors. We propose that binding of DnaK to region C is central to a conserved regulatory mechanism allowing the sensing of stress by the heat shock gene transcription machinery.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Bactérias/genética , Sítios de Ligação/genética , Sequência Conservada , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The single mutation L30 K in the Hu-Yap65 WW domain increased the stability of the complex with the peptide GTPPPPYTVG (K(d)=40(+/-5) microM). Here we report the refined solution structure of this complex by NMR spectroscopy and further derived structure-activity relationships by using ligand peptide libraries with truncated sequences and a substitution analysis that yielded acetyl-PPPPY as the smallest high-affinity binding peptide (K(d)=60 microM). The structures of two new complexes with weaker binding ligands chosen based on these results (N-(n-octyl)-GPPPYNH(2) and Ac-PLPPY) comprising the wild-type WW domain of Hu-Yap65 were determined. Comparison of the structures of the three complexes were useful for identifying the molecular basis of high-affinity: hydrophobic and specific interactions between the side-chains of Y28 and W39 and P5' and P4', respectively, and hydrogen bonds between T37 (donnor) and P5' (acceptor) and between W39 (donnor) and T2' (acceptor) stabilize the complex.The structure of the complex L30 K Hu-Yap65 WW domain/GTPPPPYTVG is compared to the published crystal structure of the dystrophin WW domain bound to a segment of the beta-dystroglycan protein and to the solution structure of the first Nedd4 WW domain and its prolin-rich ligand, suggesting that WW sequences bind proline-rich peptides in an evolutionary conserved fashion. The position equivalent to T22 in the Hu-Yap65 WW domain sequence is seen as responsible for differentiation in the binding mode among the WW domains of group I.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Substituição de Aminoácidos/genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/genética , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Alinhamento de Sequência , Soluções , Termodinâmica , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
The monoclonal antibody tAb2 binds the N-terminal sequence of transforming growth factor alpha, VVSHFND. With the help of combinatorial peptide libraries it is possible to find homologous peptides that bind tAb2 with an affinity similar to that of the epitope. The conformational flexibility of short peptides can be constrained by cyclization in order to improve their affinity to the antibody and their stability towards proteolysis. Two cyclic peptides which are cross-reactive binders for tAb2 were selected earlier using combinatorial peptide libraries. One is cyclized by an amide bond between the N-alpha group and the side-chain of the last residue (cyclo-SHFNEYE), and the other by a disulfide bridge (cyclo-CSHFNDYC). The complex structures of tAb2 with the linear epitope peptide VVSHFND and with cyclo-SHFNEYE were determined by X-ray diffraction. Both peptides show a similar conformation and binding pattern in the complex. The linear peptide SHFNEYE does not bind tAb2, but cyclo-SHFNEYE is stabilized in a loop conformation suitable for binding. Hence the cyclization counteracts the exchange of aspartate in the epitope sequence to glutamate. Isothermal titration calorimetry was used to characterize the binding energetics of tAb2 with the two cyclic peptides and the epitope peptide. The binding reactions are enthalpically driven with an unfavorable entropic contribution under all measured conditions. The association reactions are characterized by negative DeltaC(p) changes and by the uptake of one proton per binding site. A putative candidate for proton uptake during binding is the histidine residue in each of the peptides. Hydrogen bonds and the putative formation of an electrostatic pair between the protonated histidine and a carboxy group may contribute markedly to the favorable enthalpy of complex formation. Implications to cyclization of peptides for stabilization are discussed.
Assuntos
Anticorpos Monoclonais/imunologia , Reações Cruzadas/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/imunologia , Fator de Crescimento Transformador alfa/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Sequência de Bases , Sítios de Ligação de Anticorpos , Calorimetria , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/imunologia , Cristalografia por Raios X , Entropia , Epitopos/química , Epitopos/imunologia , Histidina/química , Histidina/metabolismo , Ligação de Hidrogênio , Fragmentos Fab das Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/imunologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Eletricidade Estática , Fator de Crescimento Transformador alfa/química , Água/química , Água/metabolismoRESUMO
The knowledge (antigen-derived peptide scans)- and library (de novo)-based mapping of linear and discontinuous antibody epitopes as well as protein-protein contact sites in general by spot synthesis now is a well established technique. Due to its automation, this technique also promises great potential for applications in functional genomics. It should help to elucidate the complex network of interacting protein molecules involved in signal transduction events (Adam-klages et al. 1996; Hoffmüller et al. 1999). Although little chemistry is involved in the preparation of peptide scans or libraries and the synthesis procedure is relatively simple, the laboratories of immunologists or molecular biologists are often not equipped to perform spot synthesis. In this case scans or libraries can be purchased from commercial suppliers.
Assuntos
Mapeamento de Epitopos/métodos , Proteínas/metabolismo , Reações Antígeno-Anticorpo , Antígenos/imunologia , Sítios de Ligação , Epitopos de Linfócito B/imunologia , Humanos , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/imunologiaRESUMO
The transcription factor STAT5 is activated by multiple hematopoietic cytokine receptors and has been implicated in the induction of cellular processes such as differentiation, proliferation and antiapoptotic activities. Here, we report cloning of the cDNA and characterization of a mutant STAT5a protein that is expressed in interleukin-3 (IL-3)-independently growing FDCP-1 cells. Analysis of the cDNA revealed a deletion of both the transactivation and the SH2 domains. Stable expression of the protein in parental IL-3-dependent cells results in elevated DNA binding activity of wild type (WT)-STAT5 in the nucleus, enhanced growth rates and a reduced susceptibility to undergo apoptosis after withdrawal of IL-3. Although the protein is not present in DNA/protein complexes in the nucleus, we observed pronounced effects on IL-3-induced signal transduction. The results suggest competition of the mutant protein with cytosolic mechanisms regulating STAT5 activity. In conclusion, the data support the hypothesis of an involvement of STAT5 in mitogenic and antiapoptotic signaling.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite , Deleção de Sequência/genética , Transativadores/genética , Transativadores/metabolismo , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Genes Reporter/genética , Interleucina-3/farmacologia , Dados de Sequência Molecular , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores da Eritropoetina/química , Fator de Transcrição STAT5 , Transdução de Sinais/efeitos dos fármacos , Transativadores/química , Ativação Transcricional/efeitos dos fármacos , Transfecção , Domínios de Homologia de srcRESUMO
The growing range of applications for peptide arrays synthesized on coherent membranes by the SPOT-synthesis method proves they have emerged as a powerful proteomics technique to study molecular recognition events and identify biologically active peptides. Several developments, such as the introduction of novel polymeric surfaces, linkers, synthesis/cleavage strategies and detection methods, are facilitating an increasing spectrum of accessible compounds and applications in biological or pharmaceutical research.
Assuntos
Técnicas de Química Combinatória , Peptídeos , Linfócitos B/imunologia , Biotecnologia/métodos , Mapeamento de Epitopos , Linfócitos T/imunologiaRESUMO
We provide a general route to fingerprint analyses of peptide-antibody interactions using a novel chemically synthesized peptide library. A combinatorial clustered amino acid peptide library XO1O2O3O4X (O = one of six amino acid clusters [APG], [DE], [HKR], [NQST], [FYW] and [ILVM]; X = randomized position) bound to a continuous cellulose membrane support was designed to overcome the problem of combinatorial explosion in the synthesis of peptide libraries. This library served as the starting point for the identification and detailed characterization of a TGF alpha peptide epitope recognized by the antibody Tab2. By analysing 1728 hexapeptide mixtures and 1600 single hexapeptides we identified a large number of structurally different high affinity Tab2 binding molecules. Our data provide a detailed picture of the structural basis of this antibody-peptide interaction. In addition to the detection of key amino acids involved in Tab2 binding we observed a high variability of Tab2 binding sequences supporting an induced fit mechanism in antibody-peptide recognition. In contrast, a phage display hexapeptide library led to the detection of only one dominant Tab2 binding peptide. The data obtained also demonstrate the influence of phage proteins on the interaction between the antibody and the displayed peptide. Comparing both approaches with regard to ease of handling and identified sequences, the chemical libraries are clearly favored to study antibody-peptide interactions.
Assuntos
Anticorpos , Oligopeptídeos/química , Oligopeptídeos/imunologia , Mapeamento de Peptídeos/métodos , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Epitopos/química , Epitopos/genética , Inovirus/genética , Dados de Sequência Molecular , Oligopeptídeos/genéticaRESUMO
Peptides derived from the CDRs of the anti-TNF alpha monoclonal antibody Di62 were tested for inhibition of binding of Di62 to TNF alpha as well as of TNF alpha to its 55 and 75 kDa receptor. A peptide derived from the CDR1 of the light chain was shown to specifically inhibit Di62 binding to TNF alpha with markedly higher activity (Ki = 4 microM) than all other CDR-derived peptides. This peptide also significantly inhibited binding of TNF alpha to its 55 and 75 kDa receptor and protected L929 cells from the cytotoxic effect of TNF alpha (IC50 = 6 microM). The C-terminal region of this peptide, which is homologous to the 55 and 75 kDa TNF receptor, was found to be essential for activity.
Assuntos
Anticorpos Monoclonais/imunologia , Região Variável de Imunoglobulina/imunologia , Fator de Necrose Tumoral alfa/imunologia , Sequência de Aminoácidos , Animais , Ligação Competitiva/imunologia , Testes Imunológicos de Citotoxicidade , Ensaio de Imunoadsorção Enzimática , Células L , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Radioimunoensaio , Receptores do Fator de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
The interaction of a murine monoclonal antibody (CB 4-1) against the core protein p24 of HIV-1 with its peptide antigen was studied in detail. The amino acid sequence of the variable regions of the heavy and light chain as derived from DNA sequencing was used to model the structure of the antigen binding region on the basis of reported Fab structures from the Brookhaven Protein Data Base. A linear peptide epitope responsible for the p24 binding to the antibody was determined by peptide scan. Subsequent N- and C-terminal truncation of the corresponding sequence region as well as amino acid substitutions were performed to recognize the epitope and the amino acid residues critical for antibody binding. These data were used to derive a structural model of the peptide-antibody interaction.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Proteína do Núcleo p24 do HIV/imunologia , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo , Simulação por Computador , Ligação de Hidrogênio , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/imunologia , Modelos Moleculares , Dados de Sequência MolecularRESUMO
Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.