RESUMEN
Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Epítopos , Subunidad alfa1 del Receptor de Interleucina-13/química , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Sitios de Unión/inmunología , Cristalografía por Rayos X , Dimerización , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Interleucina-13/inmunología , Interleucina-13/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Leucina/metabolismo , Ratones , Ratones Transgénicos , Estructura Terciaria de ProteínaRESUMEN
A number of secreted cytokines, such as interleukin-6 (IL-6), are attractive targets for the treatment of inflammatory diseases. We have determined the solution structure of mouse IL-6 to assess the functional significance of apparent differences in the receptor interaction sites (IL-6Rα and gp130) suggested by the fairly low degree of sequence similarity with human IL-6. Structure-based sequence alignment of mouse IL-6 and human IL-6 revealed surprising differences in the conservation of the two distinct gp130 binding sites (IIa and IIIa), which suggests a primacy for site III-mediated interactions in driving initial assembly of the IL-6/IL-6Rα/gp130 ternary complex. This is further supported by a series of direct binding experiments, which clearly demonstrate a high affinity IL-6/IL-6Rα-gp130 interaction via site III but only weak binding via site II. Collectively, our findings suggest a pathway for the evolution of the hexameric, IL-6/IL-6Rα/gp130 signaling complex and strategies for therapeutic targeting. We propose that the signaling complex originally involved specific interactions between IL-6 and IL-6Rα (site I) and between the D1 domain of gp130 and IL-6/IL-6Rα (site III), with the later inclusion of interactions between the D2 and D3 domains of gp130 and IL-6/IL-6Rα (site II) through serendipity. It seems likely that IL-6 signaling benefited from the evolution of a multipurpose, nonspecific protein interaction surface on gp130, now known as the cytokine binding homology region (site II contact surface), which fortuitously contributes to stabilization of the IL-6/IL-6Rα/gp130 signaling complex.
Asunto(s)
Receptor gp130 de Citocinas/química , Evolución Molecular , Interleucina-6/química , Complejos Multiproteicos/química , Receptores de Interleucina-6/química , Animales , Sitios de Unión , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/metabolismo , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/terapia , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mapeo Peptídico/métodos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Alineación de SecuenciaRESUMEN
The involvement of elongation factor-2 (EEF-2), a key-protein of peptide-chain elongation, in the slowing down of protein synthesis during cardiac ageing was addressed. EEF-2 was measured in rat heart extracts and isolated rat cardiomyocytes (CM) from newborn and adult rats using sodium-dodecylsulphate polyacrylamide gel electrophoresis after specific labeling with [32P]ADP-ribosylation or immunoblot. The age-dependent proportional content of several eucaryotic elongation factor-2 (eEF-2) subtypes in rat CM and rat heart extracts was compared using one-dimensional isoelectric focusing. EEF-2 was considerably reduced in the hearts of adult compared to neonatal rats (P<0.01). EEF-2 was also significantly decreased in isolated CM from adult versus newborn rats and during prolonged cultivation of neonatal CM. Cellular ageing was combined with reduced protein synthesis. During adolescence the eEF-2 variants shifted to acidic subtypes. Young adult and old rats revealed similar amounts and subtype distribution of cardiac eEF-2. Only the more acidic eEF-2 variants appeared to contain phosphorylated eEF-2. We concluded that total cardiac eEF-2 and its subtype pattern might play an important role in developmental and age-related proteomic changes.
Asunto(s)
Envejecimiento/metabolismo , Corazón/fisiología , Miocardio/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Adenosina Difosfato/metabolismo , Animales , Animales Recién Nacidos , Extractos Celulares , Células Cultivadas , Senescencia Celular , Citosol/metabolismo , Miocardio/citología , Fosforilación , Biosíntesis de Proteínas , Isoformas de Proteínas/metabolismo , Ratas , Ratas WistarRESUMEN
High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure-regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor-2 (eEF-2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF-2 decreased by 40%-50% within 12 h of the pressure treatment. However, the steady-state mRNA level of eEF-2 was not affected by the pressure. Cycloheximide addition after 4 h-pressure treatment suggested that the half-life of eEF-2 protein was shorter in pressurized cells. eEF-2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF-2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56-phosphorylated form of eEF-2 showed that phosphorylation of eEF-2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF-2. However, this inhibition may result from the decrease of cellular eEF-2 protein.