RESUMEN
Members of the IL-6/IL-12 cytokine family are critical regulators of innate and adaptive immunity and have emerged as key players controlling inflammatory and autoimmune disorders. This cytokine family comprises of IL-12, IL-23, IL-27, and IL-35, each consisting of distinct α- and ß-cytokine subunits that form heterodimers. A new member of this family, IL-39, was identified in the murine species and was shown to consist of the IL-23p19 and Epstein-Barr Virus-induced 3 (EBI3) subunits. Subsequently, it was shown that IL-39 was implicated in the immunopathogenesis of murine experimental lupus erythematosus. The existence of IL-39 in the human system has yet to be confirmed. Based on the clinical success of IL-23p19 neutralizing approaches in moderate-to-severe psoriasis, anti-IL-23p19 antibodies in the clinic may not only neutralize IL-23, but additionally IL-39, implying that IL-39 might also contribute to the pathogenesis of psoriasis. It is therefore pivotal to demonstrate IL-39 expression and to characterize its function in the human system. In this study, we provided evidence for the existence of secreted heterodimeric p19 and EBI3 complexes in supernatants originating from p19 and EBI3 transfected HEK293FT cells. We attempted to detect IL-39 expression from stimulated human primary B cells, human keratinocytes and in vitro polarized human macrophages. Whereas, the expression of p19 and EBI3 mRNA was elevated, we failed to detect p19 and EBI3 heterodimers. Functional assays were conducted with conditioned media containing human IL-39 or with a human recombinant IL-39 Fc protein. Immune cells targeted by IL-39 in mouse, such as neutrophils and PBMCs, did not respond to human IL-39 stimulation and IL-39 failed to activate STAT3 in a reporter cell line. These results suggest that, while the secretion of p19/EBI3 complexes can be forced in human cells, it is secreted below the lower quantity of detection or it has no functional role.
Asunto(s)
Inmunidad Adaptativa/genética , Inmunidad Innata/genética , Subunidad p19 de la Interleucina-23/genética , Interleucinas/genética , Antígenos de Histocompatibilidad Menor/genética , Receptores de Citocinas/genética , Animales , Humanos , Subunidad p19 de la Interleucina-23/inmunología , Interleucinas/inmunología , Ratones , Antígenos de Histocompatibilidad Menor/inmunología , Neutrófilos/inmunología , Receptores de Citocinas/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunologíaRESUMEN
Mitochondrial Ca2+ uptake depends on the mitochondrial calcium uniporter (MCU) complex, a highly selective channel of the inner mitochondrial membrane (IMM). Here, we screen a library of 44,000 non-proprietary compounds for their ability to modulate mitochondrial Ca2+ uptake. Two of them, named MCU-i4 and MCU-i11, are confirmed to reliably decrease mitochondrial Ca2+ influx. Docking simulations reveal that these molecules directly bind a specific cleft in MICU1, a key element of the MCU complex that controls channel gating. Accordingly, in MICU1-silenced or deleted cells, the inhibitory effect of the two compounds is lost. Moreover, MCU-i4 and MCU-i11 fail to inhibit mitochondrial Ca2+ uptake in cells expressing a MICU1 mutated in the critical amino acids that forge the predicted binding cleft. Finally, these compounds are tested ex vivo, revealing a primary role for mitochondrial Ca2+ uptake in muscle growth. Overall, MCU-i4 and MCU-i11 represent leading molecules for the development of MICU1-targeting drugs.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Células HeLa , Humanos , Modelos MolecularesRESUMEN
A large unmet medical need exists for safer antithrombotic drugs because all currently approved anticoagulant agents interfere with hemostasis, leading to an increased risk of bleeding. Genetic and pharmacologic evidence in humans and animals suggests that reducing factor XI (FXI) levels has the potential to effectively prevent and treat thrombosis with a minimal risk of bleeding. We generated a fully human antibody (MAA868) that binds the catalytic domain of both FXI (zymogen) and activated FXI. Our structural studies show that MAA868 traps FXI and activated FXI in an inactive, zymogen-like conformation, explaining its equally high binding affinity for both forms of the enzyme. This binding mode allows the enzyme to be neutralized before entering the coagulation process, revealing a particularly attractive anticoagulant profile of the antibody. MAA868 exhibited favorable anticoagulant activity in mice with a dose-dependent protection from carotid occlusion in a ferric chloride-induced thrombosis model. MAA868 also caused robust and sustained anticoagulant activity in cynomolgus monkeys as assessed by activated partial thromboplastin time without any evidence of bleeding. Based on these preclinical findings, we conducted a first-in-human study in healthy subjects and showed that single subcutaneous doses of MAA868 were safe and well tolerated. MAA868 resulted in dose- and time-dependent robust and sustained prolongation of activated partial thromboplastin time and FXI suppression for up to 4 weeks or longer, supporting further clinical investigation as a potential once-monthly subcutaneous anticoagulant therapy.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticoagulantes/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Factor XI/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Adolescente , Adulto , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Anticoagulantes/farmacología , Femenino , Humanos , Inmunoglobulina G/farmacología , Inmunoglobulina G/uso terapéutico , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Simulación del Acoplamiento Molecular , Trombosis/sangre , Adulto JovenRESUMEN
The design and synthesis of macrocyclic inhibitors of human rhinovirus 3C protease is described. A macrocyclic linkage of the P1 and P3 residues, and the subsequent structure-based optimization of the macrocycle conformation and size led to the identification of a potent biochemical inhibitor 10 with sub-micromolar antiviral activity.
Asunto(s)
Antivirales/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Diseño de Fármacos , Compuestos Macrocíclicos/farmacología , Rhinovirus/efectos de los fármacos , Proteínas Virales/antagonistas & inhibidores , Proteasas Virales 3C , Antivirales/síntesis química , Antivirales/química , Cristalografía por Rayos X , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Relación Dosis-Respuesta a Droga , Humanos , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación Molecular , Rhinovirus/enzimología , Relación Estructura-Actividad , Proteínas Virales/metabolismoRESUMEN
Homologues of signal peptide peptidase (SPPLs) are putative aspartic proteases that may catalyse regulated intramembrane proteolysis of type II membrane-anchored signalling factors. Here, we show that four human SPPLs are each sorted to a different compartment of the secretory pathway. We demonstrate that SPPL2a and SPPL2b, which are sorted to endosomes and the plasma membrane, respectively, are functional proteases that catalyse intramembrane cleavage of tumour necrosis factor alpha (TNFalpha). The two proteases promoted the release of the TNFalpha intracellular domain, which in turn triggers expression of the pro-inflammatory cytokine interleukin-12 by activated human dendritic cells. Our study reveals a critical function for SPPL2a and SPPL2b in the regulation of innate and adaptive immunity.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Proteínas Bacterianas/metabolismo , Células Dendríticas/efectos de los fármacos , Interleucina-12/biosíntesis , Membranas Intracelulares/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Proteínas Bacterianas/genética , Western Blotting , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Citometría de Flujo , Células HeLa , Humanos , Hidrólisis/efectos de los fármacos , Membranas Intracelulares/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Fluorescente , Interferencia de ARNRESUMEN
Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS.
Asunto(s)
Proteoma , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Cromatografía de Afinidad , ADN Complementario , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Investigación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TemperaturaRESUMEN
As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.