RESUMEN
Designing synthetic surrogates of functional proteins is an important, albeit challenging, task in the field of chemistry. A strategy toward the design of synthetic agonists for growth factor or cytokine receptors that elicit a desired signal activity has been in high demand, as such ligands hold great promise as safer and more effective therapeutics. In the present study, we used a DNA aptamer as a building block and described the strategy-guided design of a synthetic receptor agonist with fine-tuned agonism. The developed synthetic partial agonist can regulate therapeutically relevant cellular activities by eliciting fine-tuned receptor signaling.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/agonistas , Receptores de Citocinas/agonistas , Células A549 , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Movimiento Celular/efectos de los fármacos , Dimerización , Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Microscopía Fluorescente , Unión Proteica , Proteínas Proto-Oncogénicas c-met/agonistas , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Cytokines comprise a large family of secreted ligands that are critical for the regulation of immune homeostasis. Cytokines initiate signaling via dimerization or oligomerization of the cognate receptor subunits, triggering the activation of the Janus Kinases (JAKs)/ signal transducer and activator of transcription (STATs) pathway and the induction of specific gene expression programs and bioactivities. Deregulation of cytokines or their downstream signaling pathways are at the root of many human disorders including autoimmunity and cancer. Identifying and understanding the mechanistic principles that govern cytokine signaling will, therefore, be highly important in order to harness the therapeutic potential of cytokines. In this review, we will analyze how biophysical (ligand-receptor binding geometry and affinity) and cellular (receptor trafficking and intracellular abundance of signaling molecules) parameters shape the cytokine signalosome and cytokine functional pleiotropy; from the initial cytokine binding to its receptor to the degradation of the cytokine receptor complex in the proteasome and/or lysosome. We will also discuss how combining advanced protein engineering with detailed signaling and functional studies has opened promising avenues to tackle complex questions in the cytokine signaling field.
Asunto(s)
Citocinas/genética , Homeostasis/inmunología , Ingeniería de Proteínas , Receptores de Citocinas/metabolismo , Transducción de Señal/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Quinasas Janus/metabolismo , Receptores de Citocinas/agonistas , Receptores de Citocinas/inmunología , Factores de Transcripción STAT/metabolismoRESUMEN
The realisation that UV radiation (UVR) exposure could induce a suppressed immune environment for the initiation of carcinogenesis in the skin was first described more than 40 years ago. Van der Leun and his colleagues contributed to this area in the 1980s and 90s by experiments in mice involving UV wavelength and dose-dependency in the formation of such tumours, in addition to illustrating both the local and systemic effect of the UVR on the immune system. Since these early days, many aspects of the complex pathways of UV-induced immunosuppression have been studied and are outlined in this review. Although most experimental work has involved mice, it is clear that UVR also causes reduced immune responses in humans. Evidence showing the importance of the immune system in determining the risk of human skin cancers is explained, and details of how UVR exposure can down-regulate immunity in the formation and progression of such tumours reviewed. With increasing knowledge of these links and the mechanisms of UVR-induced immunosuppression, novel approaches to enhance immunity to skin tumour antigens in humans are becoming apparent which, hopefully, will reduce the burden of UVR-induced skin cancers in the future.
Asunto(s)
Tolerancia Inmunológica/efectos de la radiación , Neoplasias Cutáneas/etiología , Rayos Ultravioleta , Animales , Antígeno B7-H1/química , Antígeno B7-H1/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/efectos de la radiación , Humanos , Sistema Inmunológico/metabolismo , Sistema Inmunológico/efectos de la radiación , Neuropéptidos/metabolismo , Receptores de Citocinas/agonistas , Receptores de Citocinas/metabolismo , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismoRESUMEN
Regulatory T-Cells (Tregs) play a protective role against the development of atherosclerosis. Moreover, thymic stromal lymphopoietin (TSLP)/thymic stromal lymphopoietin receptor (TSLPR) signaling in myeloid dendritic cells (DCs) promote Treg differentiation. Here, we examined the potential role of TSLP/TSLPR on Treg homeostasis in atherosclerosis. The frequencies of both latency-associated peptide (LAP)(+) and Foxp3(+) Tregs were reduced in the thymus and spleen of ApoE(-/-) mice compared with C57BL/6 mice, and this effect was associated with decreased thymic output. The tolerogenic function of DCs obtained from ApoE(-/-) mice was compromised compared with those from C57BL/6 mice. The expression of TSLP and TSLPR was also inhibited in ApoE(-/-) mice. In addition, we found that ox-LDL attenuated TSLP expression in cultured thymic epithelial cells (TECs) through the activation of retinoid X receptor alpha (RXRA) and IL-1ß and decreased LAP and PD-L1 expression in oxLDL-activated DCs while both were up-regulated in TSLP-activated DCs. We also observed that the TSLP-DCs mediated differentiation of Tregs was abrogated through LAP neutralization. Furthermore, TSLP injection rescued Treg defects in ApoE(-/-) mice. These findings suggest that Treg defects in ApoE(-/-) mice might partially be attributed to the disruption of TSLP-TSLPR-LAP signaling in epithelial cells (ECs) and DCs.
Asunto(s)
Aterosclerosis/metabolismo , Comunicación Celular , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Epiteliales/metabolismo , Hiperlipidemias/metabolismo , Inmunoglobulinas/metabolismo , Péptidos/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal , Linfocitos T Reguladores/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Comunicación Celular/efectos de los fármacos , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Citocinas/administración & dosificación , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Factores de Transcripción Forkhead/metabolismo , Hiperlipidemias/genética , Hiperlipidemias/inmunología , Hiperlipidemias/patología , Tolerancia Inmunológica , Lipoproteínas LDL/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Péptidos/aislamiento & purificación , Receptores de Citocinas/agonistas , Transducción de Señal/efectos de los fármacos , Bazo/inmunología , Bazo/metabolismo , Linfocitos T Reguladores/inmunología , Timo/inmunología , Timo/metabolismo , Linfopoyetina del Estroma TímicoRESUMEN
ß-Arrestin, a signal adaptor protein, mediates intracellular signal transductions through protein-protein interactions by bringing two or more proteins in proximity. Extracellular signal-regulated kinase (ERK), a protein kinase in the family of mitogen-activated protein kinases (MAPKs), is involved in various receptor signal pathways. Interaction of ERK with ß-arrestin or formation of ERK/ß-arrestin signal complex occurs in response to activation of a variety of cell surface receptors. The ERK/ß-arrestin signal complex may be a common transducer to converge a variety of extracellular stimuli to similar downstream intracellular signaling pathways. By using a cell-based protein-protein interaction LinkLight assay technology, we demonstrate a direct interaction between ERK and ß-arrestin in response to extracellular stimuli, which can be sensitively and quantitatively monitored. Activations of G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and cytokine receptors promote formation of the ERK/ß-arrestin signal complex. Our data indicate that the ERK/ß-arrestin signal complex is a common transducer that participates in a variety of receptor signaling pathways. Furthermore, we demonstrate that receptor antagonists or kinase inhibitors can block the agonist-induced ERK and ß-arrestin interaction. Thus, the ERK/ß-arrestin interaction assay is useful for screening of new receptor modulators.
Asunto(s)
Arrestinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Transducción de Señal/efectos de los fármacos , Arrestinas/genética , Línea Celular , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/genética , Expresión Génica , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Humanos , Unión Proteica , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Receptores de Citocinas/agonistas , Receptores de Citocinas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , beta-ArrestinasRESUMEN
Activation of the innate immune system can enhance resistance to a variety of bacterial and viral infections. In situations where the etiological agent of disease is unknown, such as a bioterror attack, stimulation of innate immunity may be particularly useful as induced immune responses are often capable of providing protection against a broad range of pathogens. In particular, the threat of an intentional release of a highly virulent bacterial pathogen that is either intrinsically resistant to antibiotics, or has been weaponized via the introduction of antibiotic resistance, makes immunopotentiation an attractive complementary or alternative strategy to enhance resistance to bacterial biothreat agents. Francisella tularensis, Yersinia pestis, Bacillus anthracis, and Burkholderia mallei or pseudomallei can all be easily disseminated via the respiratory route and infections can result in high mortality rates. Therefore, there has been a marked increase in research on immunotherapeutics against these Tier 1 select agents over the last 10 years that will be covered in this review. In addition, immunopotentiation against non-Tier 1 select agents such as Brucella spp., and Coxiella burnetii has also been studied and will be reviewed here. In particular, we will focus on cellular targets, such as toll-like receptors (TLRs), carbohydrate receptors and cytokine receptors, which have been exploited by immunomodulatory regimens that confer broad-spectrum protection against virulent bacterial pathogens.
Asunto(s)
Carbunco/terapia , Brucelosis/terapia , Muermo/terapia , Factores Inmunológicos/uso terapéutico , Melioidosis/terapia , Peste/terapia , Fiebre Q/terapia , Tularemia/terapia , Animales , Carbunco/inmunología , Carbunco/microbiología , Brucelosis/inmunología , Brucelosis/microbiología , Expresión Génica , Muermo/inmunología , Muermo/microbiología , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunoterapia , Melioidosis/inmunología , Melioidosis/microbiología , Peste/inmunología , Peste/microbiología , Fiebre Q/inmunología , Fiebre Q/microbiología , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Citocinas/agonistas , Receptores de Citocinas/genética , Receptores de Citocinas/inmunología , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Tularemia/inmunología , Tularemia/microbiologíaRESUMEN
BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine that triggers dendritic cell-mediated T helper (Th)2 inflammatory responses through a receptor consisting of a heterodimer of the IL-7 receptor alpha (IL-7Ralpha) chain and the TSLP receptor (TSLPR), which resembles the cytokine receptor common gamma chain. Dendritic cells activated by TSLP prime development of CD4(+) T cells into Th2 cells contributing to the pathogenesis of allergic inflammation. We hypothesized that allergen exposure induces expression of TSLP and results in recruitment of TSLPR bearing cells in the cutaneous allergen-induced late-phase reaction (LPR) in atopic subjects. METHODS: Skin biopsies were obtained from atopic subjects (n = 9) at various times after cutaneous allergen challenge. In situ hybridization and immunohistochemistry were used to determine TSLP mRNA expression and to measure infiltration of TSLPR(+) DC in skin LPR. RT-PCR and flow cytometry were employed to analyse TSLPR expression on isolated blood DC. RESULTS: Allergen-induced skin TSLP expression occurred as early as 1 h after allergen challenge, whereas TSLPR(+) and CD11c(+) cells infiltrated relatively late (24-48 h). The majority of TSLPR(+) cells were DC co-expressing blood DC antigen-1 (BDCA-1) or BDCA-2. Freshly isolated blood DC expressed both TSLPR and IL-7Ralpha chains. Maturation and stimulation with TSLP or polyriboinosinic-polyribocytidylic acid in vitro upregulated the expression of both TSLPR and IL-7Ralpha chains in DC but not in chemoattractant receptor-homologous molecule expressed on Th2 cells(+) CD4(+) T cells. CONCLUSION: The data suggest that TSLP plays a role in augmenting, through DC recruitment and activation, the development of Th2-type T cells in allergic inflammation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citocinas/biosíntesis , Células Dendríticas/inmunología , Hipersensibilidad/inmunología , Receptores de Citocinas/metabolismo , Receptores de Interleucina-7/metabolismo , Adolescente , Adulto , Alérgenos/inmunología , Antígenos CD1 , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Citocinas/inmunología , Citocinas/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Glicoproteínas , Humanos , Hipersensibilidad/metabolismo , Inductores de Interferón/farmacología , Interleucina-15/farmacología , Interleucina-7/farmacología , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Poli I-C/farmacología , Receptores de Citocinas/agonistas , Receptores de Citocinas/inmunología , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Receptores de Interleucina-7/agonistas , Receptores de Interleucina-7/inmunología , Piel/inmunología , Piel/patología , Adulto Joven , Linfopoyetina del Estroma TímicoRESUMEN
Numerous important drugs target cytokines and growth factors or their receptors. Our understanding of the molecular mechanisms governing receptor activation and signaling has lagged in key areas, however, limiting drug discovery efforts to relatively few basic strategies. Recently, substantial progress has been made on several aspects of this problem. These include improved methods for establishing the mechanism of receptor activation, a clearer understanding of the biochemical basis for differential signaling by ligands that act through a common receptor, new methods for measuring the affinities of steps in receptor activation on live cells, and progress toward a systems level understanding of receptor signaling. These advances are providing a new understanding of the function of these receptors that presents opportunities for the development of improved drugs.
Asunto(s)
Receptores de Citocinas/agonistas , Receptores de Citocinas/antagonistas & inhibidores , Receptores de Factores de Crecimiento/agonistas , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Animales , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Receptores de Citocinas/metabolismo , Receptores de Factores de Crecimiento/metabolismoRESUMEN
Calcium is a second messenger for many signaling pathways in B cells, but its role as a receptor ligand has not been well characterized. However, pulses of free calcium were found to cause the rapid release of internal calcium stores in normal human B cells. This response appeared to be mediated by a cell surface protein with receptor properties as it could be blocked by pretreatment with trypsin and with kinase and phospholipase Cgamma inhibitors. The calcium receptor on B cells was not the conventional calcium-sensing receptor (CaSR) since B cells did not express CaSR and calcium-induced responses could not be blocked by specific CaSR inhibitors. B-cell responses to extracellular calcium activated phosphoinositide-3 kinase/AKT, calcineurin, extracellular signal regulated kinase, p38 mitogen-activated protein kinase, protein kinase C, Ca(2+)/calmodulin kinase II, and nuclear factor-kappaB signaling pathways, and resulted in transcription of the early response gene, CD83. This extracellular calcium sensor enhanced B-cell responses to Toll-like receptor, B-cell receptor, and cytokine receptor agonists. These findings suggest a means by which B cells prepare to engage in immune responses by responding to calcium fluctuations in their environment.
Asunto(s)
Linfocitos B/inmunología , Señalización del Calcio/inmunología , Calcio/inmunología , Activación de Linfocitos/inmunología , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Linfocitos B/enzimología , Calcineurina/inmunología , Calcineurina/metabolismo , Calcio/metabolismo , Humanos , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/inmunología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/inmunología , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosfolipasa C gamma/inmunología , Fosfolipasa C gamma/metabolismo , Fosfotransferasas/inmunología , Fosfotransferasas/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores Sensibles al Calcio/antagonistas & inhibidores , Receptores Sensibles al Calcio/inmunología , Receptores Sensibles al Calcio/metabolismo , Receptores de Citocinas/agonistas , Receptores de Citocinas/inmunología , Receptores de Citocinas/metabolismo , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunología , Antígeno CD83RESUMEN
The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.
Asunto(s)
Janus Quinasa 2/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Citocinas/metabolismo , Transducción de Señal , Sustitución de Aminoácidos , Animales , Línea Celular , Citocinas/metabolismo , Humanos , Janus Quinasa 2/genética , Ligandos , Ratones , Ratones Transgénicos , Mutación Missense , Fosforilación , Unión Proteica/genética , Procesamiento Proteico-Postraduccional/genética , Estructura Terciaria de Proteína/genética , Receptores de Citocinas/agonistas , Transducción de Señal/genéticaRESUMEN
NIP-004 is a novel synthetic compound developed to display human thrombopoietin (TPO) receptor (c-Mpl) agonist activity. NIP-004 displays species specificity, stimulating proliferation or differentiation of human c-Mpl-expressing cells such as UT-7/TPO and human CD34(+) cells but not murine c-Mpl-expressing cells or cynomolgus monkey cells. To test the mechanism of its action, we constructed mutant forms of c-Mpl; murine c-Mpl(L490H) dis-played a response to NIP-004, whereas human c-Mpl(H499L) lost this response, indicating that histidine in the transmembrane domain of c-Mpl is essential for its activity. Because histidine is not present in the c-Mpl transmembrane domain of rats, hamsters, rhesus macaques, and cynomolgus monkeys, we examined the in vivo efficacy of NIP-004 using mice that received xenotransplants. In immunodeficient nonobese diabetic (NOD)/Shi-scid, IL-2Rgamma(null) (NOG) mice receiving transplants of umbilical cord blood-derived CD34(+) cells, NIP-004 increased human megakaryoblasts, mature megakaryocytes, and circulating human platelets 6-fold, the latter being morphologically and functionally indistinguishable from normal human platelets. These observations indicate that NIP-004 is a novel human c-Mpl activator and induces human thrombopoiesis.
Asunto(s)
Proteínas Proto-Oncogénicas/agonistas , Receptores de Citocinas/agonistas , Trombopoyesis/efectos de los fármacos , Animales , Plaquetas/citología , Recuento de Células , Línea Celular Tumoral , Trasplante de Células Madre de Sangre del Cordón Umbilical , Supervivencia de Injerto , Histidina , Humanos , Megacariocitos/citología , Ratones , Ratones SCID , Mutación , Proteínas Proto-Oncogénicas/genética , Receptores de Citocinas/genética , Receptores de Trombopoyetina , Trasplante HeterólogoRESUMEN
A novel benzimidazole series of small-molecule thrombopoietin receptor agonists has been discovered. Herein, we discuss the preliminary exploration of structure-activity relationships within this chemotype.
Asunto(s)
Bencimidazoles/química , Bencimidazoles/farmacología , Receptores de Citocinas/agonistas , Receptores de Citocinas/metabolismo , Trombopoyetina/metabolismo , Ácidos/química , Bencimidazoles/síntesis química , Estructura Molecular , Naftoles/química , Relación Estructura-ActividadRESUMEN
OBJECTIVE: Peptide and other small molecule agonists have been described for several cytokines and growth factors. Hydrazone compounds described here as thrombopoietin receptor agonists were identified as activating STAT proteins in a Tpo responsive cell line. METHODS: STAT activation and analysis of signal transduction pathways in cell lines and normal human platelets was elucidated by Western blot and electrophoretic mobility shift assays. Proliferation assays in cell types responsive to other cytokines determined specificity for Tpo receptor. Flow cytometry quantified differentiation of CD34(+) cells into CD41(+) megakaryocytes and platelet production in vitro. RESULTS: Activation of STAT5, mitogen-activated protein kinase, p38, and early response genes by SB 394725 was similar to that induced by Tpo. SB 394725 induced a reporter gene response under a STAT activation promoter as well as the megakaryocyte-specific gpIIb promoter. The compound induced proliferation of Tpo responsive lines but demonstrated no activity in cell lines responding to other cytokines, i.e., erythropoietin, granulocyte-colony stimulating factor, interleukin-3, interferon-gamma. The response of normal human Tpo receptors was elucidated by measuring growth and differentiation of human bone marrow in vitro. Activation of endogenous Tpo receptors by SB 394725 was demonstrated in human and chimp platelets, but not in platelets of other species including mouse, dog, rabbit, or cynomolgus monkey. CONCLUSIONS: SB 394725, a small molecule with a molecular weight of 452 Da, is capable of activating Tpo-specific signal transduction, proliferation, and differentiation responses similar to the responses and functions of the protein growth factor, Tpo.
Asunto(s)
Hidrazonas/farmacología , Receptores de Citocinas/agonistas , Animales , Plaquetas , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Megacariocitos , Ratones , Proteínas de la Leche/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas Proto-Oncogénicas/agonistas , Receptores de Trombopoyetina , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Especificidad de la Especie , Transactivadores/metabolismoRESUMEN
Antibodies have brought valuable therapeutics in the clinical treatment of various diseases without serious adverse effects through their intrinsic features such as specific binding to the target antigen with high affinity, clinical safety as serum proteins, and long half-life. Agonist antibodies, furthermore, could be expected to maximize the value of therapeutic antibodies. Indeed, several IgG/IgM antibodies have been reported to induce cellular growth/differentiation and apoptosis. These agonist antibodies, however, should be further improved to exert more potent biologic activities and appropriate serum half-life depending upon the disease indications. Here, we report that IgG antibodies against the thrombopoietin receptor (Mpl), which have an absence or very weak agonist activity, can be engineered to be agonist minibodies, which include diabody or sc(Fv)2 as potent as natural ligand. Through this technological development, minibodies have been successfully constructed to bind and activate 2 types of dysfunctional mutant Mpls that cause congenital amegakaryocytic thrombocytopenia (CAMT). This drastic conversion of biologic activities by designing minibodies can be widely applicable to generate agonist minibodies for clinical application, which will constitute a new paradigm in antibody-based therapeutics.
Asunto(s)
Proteínas Portadoras/farmacología , Inmunoglobulinas/farmacología , Proteínas Oncogénicas/agonistas , Proteínas Oncogénicas/inmunología , Receptores de Citocinas/agonistas , Receptores de Citocinas/inmunología , Trombocitopenia/inmunología , Trombocitopenia/terapia , Animales , Anticuerpos Monoclonales , Autoanticuerpos/inmunología , Línea Celular Tumoral , Humanos , Inmunización , Leucemia Megacarioblástica Aguda , Ratones , Ratones Endogámicos MRL lpr , Receptores de Trombopoyetina , Trombopoyetina/inmunologíaRESUMEN
Soluble cytokine receptors regulate inflammatory and immune events by functioning as agonists or antagonists of cytokine signaling. As such, they act within complex receptor systems that include signaling receptors, nonsignaling decoy receptors, receptor-associated proteins, and soluble receptor antagonists. Soluble cytokine receptors can be generated by several mechanisms, which include proteolytic cleavage of receptor ectodomains, alternative splicing of mRNA transcripts, transcription of distinct genes that encode soluble cytokine-binding proteins, release of full-length receptors within the context of exosome-like vesicles, and cleavage of GPI-anchored receptors. Furthermore, the important role of soluble cytokine receptors in regulating host defense mechanisms is evidenced by viruses that encode soluble homologues of mammalian receptors and thereby evade innate host immune responses via the sequestration of essential cytokines.
Asunto(s)
Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/fisiología , Animales , Humanos , Mediadores de Inflamación/agonistas , Mediadores de Inflamación/antagonistas & inhibidores , Receptores de Citocinas/agonistas , Receptores de Citocinas/antagonistas & inhibidores , Transducción de Señal/inmunología , SolubilidadRESUMEN
Understanding the receptor activation mechanism is essential for the rational design of pharmacologically active ligand molecules. However, the activation mechanism of most cytokine receptors remains still unclear, and while agonism and antagonism have been described for ligand-mimetic peptides, there has been no report of inverse agonism that has been characterized for G protein-coupled receptors (GPCRs). To explore the activation mechanism of cytokine receptors, here we tried to investigate how agonism and antagonism could be altered by randomizing antibody variable region of an antibody/cytokine receptor chimera recognizing hen egg lysozyme (HEL) as an agonist. Based on our previous finding that the co-expression of V(H)-gp130 and V(L)-erythropoietin receptor (EpoR) chimeras transduced strict and efficient HEL-dependent cell growth signal, a V(H)-gp130 library encoding four randomized CDR2 residues was retrovirally infected to IL-3-dependent Ba/F3 cells already transfected with V(L)-EpoR. The selection without IL-3 resulted in a clonal expansion of the transduced cells, and interestingly some of which showed HEL dose-dependent growth suppression. Our results clearly indicate that agonism and antagonism of the antibody/cytokine receptor chimera can be readily switched by a subtle modification of the ligand binding domain as well as that of GPCRs, also implying the existence of inverse agonism in cytokine receptor superfamily.
Asunto(s)
Receptores de Citocinas/agonistas , Proteínas Recombinantes de Fusión/farmacología , Animales , Anticuerpos/genética , Antígenos/genética , Sitios de Unión , Western Blotting , Dimerización , Ratones , Mutación , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Recent studies have shown that disruption of Notch1 signaling in lymphocyte progenitors (LP) inhibits T cell development and promotes B cell development in the thymus. Conversely, inappropriate activation of Notch1 in LP inhibits B cell development and causes ectopic T cell development in the bone marrow. These observations imply that Notch1 activation must be spatially regulated to ensure that LP generate B cells in the bone marrow and T cells in the thymus. However, Notch ligands are expressed in both tissues. Studies in flies and worms have revealed that Notch activation is extremely sensitive to small changes in the amount of receptor or ligand expressed, and defined multiple mechanisms that limit Notch activation to discrete cells at specific times during development. Here, we describe how some of these mechanisms might regulate Notch activity in LP during the T/B lineage decision.
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Linfocitos B/inmunología , Receptores de Superficie Celular/metabolismo , Linfocitos T/inmunología , Factores de Transcripción , Animales , Linaje de la Célula , Proteínas de Drosophila , Endocitosis , Glicosilación , Inmunoglobulinas , Hormonas Juveniles/metabolismo , Ratones , N-Acetilglucosaminiltransferasas/metabolismo , Receptor Notch1 , Receptores de Superficie Celular/fisiología , Receptores de Citocinas/agonistas , Receptores de Citocinas/metabolismo , Transducción de SeñalRESUMEN
Ligand-induced dimerization of cell surface receptors has emerged as a general mechanism for the initiation of signal transduction. A number of therapeutically important receptor families are believed to be activated by this process. Recently available structural information, particularly for the erythropoietin receptor, has provided insight into the mechanism of receptor activation. These findings have also revealed important constraints on the nature of receptor-agonist complexes. The prospects of discovering small-molecule mimetics of such receptor agonists are discussed, including strategies which have led to the identification of a small number of peptide and non-peptide cytokine mimetics.
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Receptores de Citocinas/metabolismo , Animales , Dimerización , Humanos , Ligandos , Receptores de Citocinas/agonistas , Receptores de Citocinas/fisiología , Receptores de Eritropoyetina/metabolismo , Receptores de Eritropoyetina/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Heptahelical receptors mediate contractile and relaxant responses of bronchial smooth muscle cells to mediators and drugs. The activity of these receptors is constitutively regulated through the equilibrium between their inactive conformation R and their active conformation R*, coupled to G proteins. This equilibrium is shifted to R* by agonists, with a relaxant response to beta-adrenergic agonists and a contractile response to muscarinic agonists. In contrast, inverse agonists shift this equilibrium to R with, for instance, a putative relaxant response to muscarinic inverse agonists. The expression of heptahelical receptors at the plasma membrane correspond to the equilibrium between the endocytosis of membrane receptors and the synthesis of receptor mRNA and protein. Endocytosis is activated by agonists and decreased by inverse agonists. The transcriptional regulation of heptahelical receptors in bronchial smooth muscle cells is tightly controlled by pro-inflammatory cytokines. The variety of the involved control pathways might explain the spasmogen-selective hyperresponsiveness, allowing a reappraisal of the dogma of non selective bronchial hyperresponsiveness in asthma.
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Broncoconstricción/fisiología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Contracción Muscular/fisiología , Músculo Liso/fisiología , Receptores de Superficie Celular/biosíntesis , Receptores de Citocinas/biosíntesis , Agonistas Adrenérgicos beta/farmacología , Asma/fisiopatología , Bronquios/fisiología , Hiperreactividad Bronquial/fisiopatología , Broncoconstricción/efectos de los fármacos , Endocitosis , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Conformación Proteica , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/genética , Receptores de Citocinas/agonistas , Receptores de Citocinas/genéticaRESUMEN
Phenylpiperazine derivatives were synthesized as dual cytokine regulators with TNF-alpha suppressing and IL-10 augmenting activity. Lead optimization led to compound 5k having the potent regulatory activity and demonstrating remarkable protective effects against the lethal challenge of LPS in mice. suggesting that 5k would be a promising drug candidate for the treatment of TNF-alpha associated diseases including septic shock.