RESUMEN
The transcription factor NF-kappa B regulates many genes involved in proinflammatory and immune responses. The transport of NF-kappa B into the nucleus is essential for its biologic activity. We describe a novel, potent, and selective NF-kappa B inhibitor composed of a cell-permeable peptide carrying two nuclear localization sequences (NLS). This peptide blocks NF-kappa B nuclear localization, resulting in inhibition of cell surface protein expression, cytokine production, and T cell proliferation. The peptide is efficacious in vivo in a mouse septic shock model as well as a mouse model of inflammatory bowel disease, demonstrating that NF-kappa B nuclear import plays a role in these acute inflammatory disease models.
Asunto(s)
FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Señales de Localización Nuclear , Péptidos/farmacología , Choque Séptico/metabolismo , Secuencia de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , Línea Celular , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Humanos , Cadenas kappa de Inmunoglobulina/biosíntesis , Inmunosupresores/administración & dosificación , Inmunosupresores/farmacología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/prevención & control , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Señales de Localización Nuclear/efectos de los fármacos , Péptidos/administración & dosificación , Péptidos/síntesis química , Receptores de Antígenos de Linfocitos B/biosíntesis , Choque Séptico/inmunología , Choque Séptico/patología , Choque Séptico/prevención & control , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Hsc70, the constitutive form of the heat shock protein 70 family of proteins, is involved in a number of biological activities which include protein folding and molecular chaperoning. Previously, we had shown that the immunosuppressant 15-deoxyspergualin (DSG) specifically interacted with Hsc70, as well as the Hsp90 family of proteins. Although the exact binding site on Hsc70 for protein substrates is unknown, a recent study shows that the extreme C-terminal four amino acids 647EEVD650 play a role in regulating AT-Pase activity, substrate binding, and interaction with HDJ-1. These four amino acids are also found at the C-terminus of Hsp90 and may be involved in similar functions. In this study, we show that DSG binds specifically to this EEVD regulatory domain. Binding of DSG to Hsc70 did not affect its ability to bind peptides. These results suggest that in addition to the ATP binding domain, there are two additional substrate binding domains on Hsc70. DSG should provide a tool for understanding the role of the EEVD motif in biological processes.
Asunto(s)
Guanidinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Inmunosupresores/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Unión Competitiva , Bovinos , Reactivos de Enlaces Cruzados/metabolismo , Etildimetilaminopropil Carbodiimida/metabolismo , Guanidinas/farmacología , Inmunosupresores/farmacología , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica/efectos de los fármacosRESUMEN
The process of nuclear protein transport requires the interaction of several different proteins, either directly or indirectly with nuclear localization or targeting sequences (NLS). Recently, a number of karyopherins alpha, or NLS-binding proteins, have been identified. We have found that the karyopherins hSRP1 and hSRP1alpha are differentially expressed in various leukocyte cell lines and could be induced in normal human peripheral blood lymphocytes. We show that the two karyopherins bind with varied specificities in a sequence specific manner to different NLSs and that the sequence specificity is modulated by other cytosolic proteins. There was a correlation between binding of karyopherins alpha to different NLSs and their ability to be imported into the nucleus. Taken together, these data provide evidence for multiple levels of control of the nuclear import process.
Asunto(s)
Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Señales de Clasificación de Proteína/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Línea Celular , Humanos , Leucocitos/metabolismo , Linfocitos/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Fracciones Subcelulares/metabolismo , alfa CarioferinasRESUMEN
15-Deoxyspergualin (DSG) is an immunosuppressive agent currently in Phase I/II clinical trials. We have previously shown that DSG specifically interacts with Hsc70, a member of the heat shock protein 70 family. In this article we show that DSG appears to bind either kinetically or to a different site on Hsc70 from that of peptides. In addition, we show that DSG inhibits the localization of Hsp70 into the nucleus in response to heat shock. Finally, data are presented showing that there is a correlation between decreases in the transcription factor nuclear factor kappa B and kappa light chain expression in response to varying doses of DSG.
Asunto(s)
Guanidinas/farmacología , Inmunosupresores/farmacología , Secuencia de Aminoácidos , Animales , Unión Competitiva , Núcleo Celular/efectos de los fármacos , Guanidinas/química , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/química , Cadenas kappa de Inmunoglobulina/efectos de los fármacos , Inmunosupresores/química , Leucemia L1210 , Ratones , Datos de Secuencia Molecular , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , Péptidos/química , Unión ProteicaRESUMEN
CD28 and CTLA-4, T cell receptors for B7-1 (CD80) and B7-2 (CD86) molecules on antigen-presenting cells, transmit costimulatory signals important for optimal T cell activation. Despite sharing sequence homology and common ligands, these receptors have distinct binding properties and patterns of expression. The function of CTLA-4 during T cell activation is not well understood, although an important role is suggested by complete amino acid sequence conservation of its cytoplasmic tail in all species studied to date. We report here a role of the cytoplasmic tail of CTLA-4 in regulating its subcellular localization and cell surface expression. In activated human peripheral blood T cells, or in several transfected or transduced cell types, CTLA-4 is not primarily a cell surface protein, but rather is localized intracellularly in a region which overlaps the Golgi apparatus. Transfer of 11 cytoplasmic residues, 161TTGVYVKMPPT, from the CTLA-4 cytoplasmic tail to the homologous position in CD28 was sufficient to confer intracellular localization. Mutation of the tyrosine residue (Tyr165) in this motif to phenylalanine resulted in increased surface expression of CTLA-4. Thus, the subcellular localization of CTLA-4 is controlled by a tyrosine-containing motif within its cytoplasmic domain. Contained within this motif is a binding site for SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación/análisis , Antígeno B7-1/metabolismo , Inmunoconjugados , Glicoproteínas de Membrana/metabolismo , Abatacept , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación/química , Antígenos de Diferenciación/genética , Antígeno B7-2 , Antígenos CD28/análisis , Antígeno CTLA-4 , Células Cultivadas , Humanos , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisisRESUMEN
The effect of interferon-gamma (IFN-gamma) on the ability of human monocytic cells to process exogenous (major histocompability complex class II) antigens was investigated. The processing (i.e. protein degradation) of antigens that were internalized via Fc gamma receptor (Fc gamma R) was followed for various times after treatment of cells with IFN-gamma. THP-1 cells that had been treated with IFN-gamma for 4 h degraded antigen, internalized as an immune complex, at an enhanced rate. After 24 h of IFN-gamma treatment the rate of processing was similar to untreated cells. Unexpectedly, in cells which had been treated for 48-72 h there was a significant decrease in the rate of processing of the exogenous antigen. These effects were not due to changes in the rate of internalization of immune complex. The inhibition of the rate of processing was independent of the type of antigen, was dependent on the dose of IFN-gamma, and also occurred with normal human peripheral monocytes. Analysis of the degraded peptides by high-pressure liquid chromatography indicated that some of the peptides generated in the IFN-gamma-treated cells were both quantitatively and qualitatively different from the peptides generated in untreated cells. These data suggest that IFN-gamma modulates the way in which antigens, internalized through Fc receptors as immune complexes, are processed. Additionally, the results imply that decreases in the rate of antigen processing may lead to more efficient antigen presentation.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Antígenos/metabolismo , Interferón gamma/farmacología , Monocitos/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Línea Celular , Antígenos HLA/metabolismo , Humanos , Técnicas In Vitro , Cinética , Mapeo Peptídico , Receptores de IgG/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/inmunologíaRESUMEN
15-deoxyspergualin (DSG) is a potent immunosuppressive compound currently in clinical trials. In this study, we have characterized the uptake and intracellular localization of DSG in human peripheral blood lymphocytes (PBL's). DSG is transported into human PBL's and reaches an estimated maximum concentration of approximately 500 microM in 6 hours. The majority of the [3H]-DSG remains in the cytoplasm of cells and that which is associated with the nucleus is only loosely associated. DSG was transported by HeLa cells, as well, suggesting uptake is not specific for hematopoietic cells. Positively charged amino acids and polyamines, which are structurally similar to DSG, were unable to compete for DSG transport suggesting that DSG is transported into cells via a pathway distinct from amino acids or polyamines.
Asunto(s)
Guanidinas/sangre , Inmunosupresores/sangre , Linfocitos/metabolismo , Transporte Biológico/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Humanos , Cinética , Putrescina/farmacología , Espermidina/farmacología , Espermina/farmacología , Fracciones Subcelulares/metabolismo , TritioRESUMEN
Many monoclonal antibody-drug immunoconjugates have been evaluated for their ability to deliver cytotoxic drugs to tumors. It is essential to establish that the ability of the conjugates to bind antigen, i.e. their immunoreactivity, is not adversely affected by the drug conjugation procedure. We have described herein a measurement of the immunoreactivity of BR96-DOX, a conjugate comprised of BR96, a chimeric monoclonal antibody specific for the Le(y) tetrasaccharide commonly expressed on human carcinomas, and doxorubicin, an anticancer agent in widespread clinical use. We have employed a competitive RIA, in which microtiter wells were coated with synthetic Le(y) conjugated to human serum albumin and then incubated with 125I-labeled antibody BR96 in the presence of test conjugate or intact BR96 mAb. The test conjugates were found to compete as effectively as unconjugated BR96. This assay is highly applicable to QC processes with the intra-assay CV = 2.0% and the interassay CV = 4.3%.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Doxorrubicina/metabolismo , Antígeno Lewis X/inmunología , Afinidad de Anticuerpos , Células Cultivadas , Citometría de Flujo , Humanos , RadioinmunoensayoRESUMEN
A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Proteínas Tirosina Quinasas/análisis , Adenosina Trifosfato/metabolismo , Alcaloides/farmacología , Anticuerpos/inmunología , Autoanálisis , Unión Competitiva , Carbazoles/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Receptores ErbB/fisiología , Alcaloides Indólicos , Muramidasa/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Radioisótopos de Fósforo , Fosforilación , Fosfotirosina , Proteínas Tirosina Quinasas/inmunología , Estaurosporina , Especificidad por Sustrato/efectos de los fármacos , Tirosina/análogos & derivados , Tirosina/inmunologíaRESUMEN
The rate of nucleoside transport decreased profoundly in human promyelocytic leukemia HL-60 cells after myeloid differentiation was induced by 5-6 days of exposure to 0.8% N,N-dimethylformamide (DMF). The facilitated diffusion of 100 microM radiolabeled adenosine and 2'-deoxyadenosine, measured by rapid transport assays, decreased 10- to 20-fold. The transport of 2 microM coformycin or 2'-deoxycoformycin, which is mediated by the same mechanism and was monitored by the adenosine deaminase titration assay, decreased 29-fold. The reduction in nucleoside transport capacity after DMF treatment was confirmed by a 19-fold decrease in the number of specific binding sites per cell (from 24-30 X 10(4) to 1.2-1.7 X 10(4)) for [3H]-6-p-nitrobenzylthioinosine, a nucleoside transport inhibitor. The binding affinity of 6-p-nitrobenzylthioinosine was not altered significantly and nucleoside transport remained sensitive to the transport inhibitors, 6-p-nitrobenzylthioinosine, dipyridamole, and dilazep after DMF-induced maturation. Time-dependence studies showed that the rate of 100 microM deoxyadenosine transport was unchanged for the first 24 h of exposure to DMF but fell to about 36% of control rates at 24-26 h and then gradually decreased further to about 4-5% of control rates after 5-6 days. In contrast, transport rates of the purine bases were reduced only 2- to 3-fold in HL-60 cells after 5 days of DMF treatment. The rates of adenosine and deoxyadenosine transport were unchanged or reduced by no more than 2-fold after 5-6 days of exposure to 0.8% DMF in the following human tumor cell lines that are not inducible with DMF: ARH-77 (multiple myeloma), KG-1 (acute myelogenous), and K-562 (chronic myelogenous). Thus, changes in nucleoside transport may serve as an early, membrane-associated marker of differentiation of the HL-60 cell line.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Nucleósidos/metabolismo , Adenosina/metabolismo , Inhibidores de la Adenosina Desaminasa , Diferenciación Celular/efectos de los fármacos , Línea Celular , Coformicina/análogos & derivados , Coformicina/metabolismo , Desoxiadenosinas/metabolismo , Dimetilformamida/farmacología , Humanos , Leucemia Mieloide Aguda/patología , Pentostatina , Purinas/metabolismo , Tioinosina/análogos & derivados , Tioinosina/metabolismoRESUMEN
The effect of topical application of PGE on induction of ODC in mouse epidermis was measured. When direct induction of ODC by TPA was blocked by also applying indomethacin, maximum ODC activity occurred only when PGE was applied simultaneously with TPA 4 1/2 hr before killing of the mice. If either TPA or PGE was applied at other times, ODC activity decreased substantially. Induction of ODC by mezerein was blocked by indomethacin but restored by PGE, as was observed with TPA, but induction by ethyl phenylpropiolate was not affected by indomethacin or PGE. DMBA did not cause a consistent increase in ODC activity, nor was its inductive action affected by indomethacin or PGE. However, another weak inducer, acetic acid, exhibited elevated ODC activity when PGE was also applied. Inhibition by topical retinoic acid of ODC induction by TPA was partially overcome in a dose-response fashion by PGE. The results indicate that at least 2 events, elevation of PGE and another independent event, are required for induction of ODC activity. It appears that TPA causes at least 4 independent events essential for tumor promotion. A model for the events in the 2-stage tumor promotion model is proposed.
Asunto(s)
Alprostadil/farmacología , Diterpenos , Ornitina Descarboxilasa/biosíntesis , Forboles/toxicidad , Acetato de Tetradecanoilforbol/toxicidad , 9,10-Dimetil-1,2-benzantraceno , Animales , Inducción Enzimática/efectos de los fármacos , Femenino , Indometacina/farmacología , Ratones , Ratones Endogámicos , Modelos Biológicos , Neoplasias/inducido químicamente , Terpenos/toxicidad , Tretinoina/farmacologíaRESUMEN
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the non-promoter mezerein both induce ornithine decarboxylase activity in mouse epidermis by a route which can be blocked by indomethacin. In two-stage tumor promotion experiments in mice with mezerein as the stage II promoter, TPA was effective as the stage I promoter whether it was applied before or after an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA). There appear to be at least 4 events in promotion, only 3 of which are caused by second stage promoters.