Interleukin-1 receptor antagonist binds to the type II interleukin-1 receptor on B cells and neutrophils.

We have examined the binding of human and rodent interleukin-1 receptor antagonist (IL-1ra) to the type II IL-1 receptor on the human B cell line, Raji, on the mouse pre-B cell line, 70Z/3, and on human polymorphonuclear leukocytes (PMNs). Human IL-1ra binds to the receptors on the human B cells with an affinity (KD = 15 +/- 3 nM) equal to that of IL-1 alpha and only 15-fold lower than that of IL-1 beta and, likewise, binds to human PMNs with an affinity (KD = 8 +/- 4 nM) 15-fold lower than that of IL-1 beta. Mouse and rat IL-1ra bind to these two human cell types with an affinity similar to that of the human protein. Human IL-1ra binds very weakly to the type II receptor on the mouse pre-B cells with an affinity (KD = 1.4 +/- 0.2 microM) about 1500-fold lower than human IL-1 beta. Mouse and rat IL-1ra also bind to the mouse pre-B cells with low affinity. The weak binding of the three IL-1ra proteins to these mouse cells appears to be more a consequence of the cell type rather than species specificity. There may be a population of cells for which the actions of IL-1 cannot be effectively opposed by IL-1ra, although this group does not include mature B cells and PMNs.     

Interleukin-1 receptor antagonist: effectiveness against interleukin-1 fever.

Conscious cats were used to examine the effectiveness of the interleukin-1 receptor antagonist against the fever induced by interleukin-1 and endotoxin. Although inactive by itself, the antagonist (three 1-micrograms bolus injections at 10-min intervals), injected into the third ventricle, attenuated the febrile response to a subsequent intracerebroventricular bolus of interleukin-1. The rise in prostaglandin E2 levels in cerebrospinal fluid, which is a characteristic feature of fever, was curtailed as well. The interleukin-1 antagonist had little or no inhibitory effect on the response to an intracerebroventricular bolus of endotoxin, even though a higher dose was employed (2-micrograms bolus injections given three times at 10-min intervals and six times at 30-min intervals, respectively, before and after endotoxin administration). At either dosage, the intracerebroventricular antagonist was completely ineffective against an intravenous bolus injection of interleukin-1 or endotoxin and both fever and prostaglandin E2 elevation developed unabated. We conclude that brain receptors mediating the pyrogenic action of centrally injected interleukin-1 are susceptible to the antagonist. The same receptors, however, are seemingly not activated by systemic pyrogens. Our findings are consistent with the concept of circulating interleukin-1 acting outside the blood-brain barrier in the normal sequence of fever.     

Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist

Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using high-resolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANS-protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25 degrees C and 37 degrees C. Overall, the affinity of ANS was lower at 37 degrees C compared to 25 degrees C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solvent-exposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation.     

Anion binding and controlled aggregation of human interleukin-1 receptor antagonist

Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway.     

HSP70 inhibits stress-induced cardiomyocyte apoptosis by competitively binding to FAF1

Stress-induced cardiomyocyte apoptosis plays an important role in the pathogenesis of a variety of cardiovascular diseases. Our early studies showed that HSP70 effectively inhibited apoptosis, but the underlying mechanism remained unclear. Fas-associated factor 1 (FAF1) is a member of the Fas death-inducing signaling complex (Fas-DISC) that acts upstream of caspase-8. We investigated the interactions among FAF1, HSP70, and FAS in stressed cardiomyocytes to elucidate the protective mechanism of HSP70. FAS and caspase-3/8 activity was higher in cardiomyocytes undergoing stress-induced apoptosis in restraint-stressed rats compared with cardiomyocytes in non-stressed rats, which indicated that the Fas signaling pathway was activated after restraint stress. Geranylgeranylacetone (GGA) induced an increase in HSP70 expression, which reduced stress-induced apoptosis. Additionally, overexpression of HSP70 via transfection with the pEGFP-rHSP70 plasmid attenuated norepinephrine (NE)-induced apoptosis. FAF1 expression increased during stress-induced apoptosis, and overexpression of FAF1 exacerbated NE-induced apoptosis. We also found that HSP70 interacted with FAF1. Overexpression of HSP70 inhibited the binding of FAF1 to FAS in H9C2 cells, which indicated that HSP70 suppressed NE-induced apoptosis by competitively binding to FAF1. An N-terminal deletion mutant of HSP70 (HSP70-△N) was unable to interact with FAF1. After HSP70-△N was transfected into H9C2 cells, the cells were unable to attenuate the NE-induced increases in caspase-8 and apoptosis. These results indicate that the 1–120 sequence of HSP70 binds to FAF1, which alters the interactions between FAS and FAF1 and inhibits the activation of the Fas signaling pathway and apoptosis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0589-9) contains supplementary material, which is available to authorized users.     

Microbial polysaccharide, HS-142-1, competitively and selectively inhibits ANP binding to its guanylyl cyclase-containing receptor.

During the search for ANP receptor ligands of microbial origin, we isolated a novel polysaccharide, HS-142-1, from culture broth of Aureobasidium sp. HS-142-1 inhibited [125I]-rANP binding to ANP receptor in rabbit kidney cortex membranes with an IC50 of 0.3 mu g/ml, but gave no effects on specific binding of [125I]-Endothelin nor [125I]-Angiotensin II to their respective receptors in bovine lung membranes. HS-142-1 competitively and selectively inhibited ANP binding to its guanylyl cyclase-containing receptor purified from solubilized bovine adrenocortical membranes and blocked cGMP production elicited by ANP. HS-142-1 is the first non-peptide antagonist selective for ANP functional receptor and will be a powerful tool to elucidate the physiological functions of ANP.     

X-ray crystal structure of a small antagonist peptide bound to interleukin-1 receptor type 1

Interleukin (IL-1)alpha and IL-1beta are important mediators of inflammation. The binding of IL-1 to interleukin-1 receptor (IL-1R) type 1 is the initial step in IL-1 signal transduction and therefore is a tempting target for anti-inflammatory therapeutics. To advance our understanding of IL-1R1 binding interactions, we have determined the structure of the extracellular domains of IL-1R1 bound to a 21-amino acid IL-1 antagonist peptide at 3.0-A resolution. The antagonist peptide binds to the domain 1/2 junction of the receptor, which is a conserved binding site for IL-1beta and IL-1 receptor antagonist (IL-1ra). This co-crystal structure also reveals that considerable flexibility is present in IL-1R1 because the carboxyl-terminal domain of the receptor is rotated almost 170 degrees relative to the first two domains of the receptor compared with the previously solved IL-1R1.ligand structures. The structure shows an unexpected binding mode for the peptide and may contribute to the design of smaller IL-1R antagonists.     

Platelet factor 4 selectively inhibits binding of TGF-beta 1 to the type I TGF-beta 1 receptor.

A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monotonicity of interleukin-1 receptor-ligand binding with respect to antagonist in the presence of decoy receptor

We consider the interaction between interleukin-1 IL-1, its receptor IL-1RI, the receptor antagonist IL-1Ra and a decoy receptor (or trap) that binds both with the ligand and the antagonist. We study how the interaction between IL-1Ra and the decoy receptor influences the effect of either reagent on reducing the equilibrium concentration of the receptor-ligand complex. We obtain that, given a certain relationship among the equilibrium constants and the total concentrations of solutes, IL-1Ra can reverse the effect of the decoy receptor of decreasing the equilibrium concentration of the receptor-ligand complex. This finding derives from a mathematical result applicable to any reversible chemical reaction system comprising four species arranged in a square such that each species binds its two immediate neighbors. The result gives the monotonicity of the equilibrium concentrations of the complex species as functions of the total concentrations of the simple species.     

Evidence that neomycin inhibits binding of herpes simplex virus type 1 to the cellular receptor.

The effect of neomycin, a phosphoinositide-binding aminoglycoside, on herpes simplex virus type 1 (HSV-1) infection of BHK cells was studied. We showed earlier that it specifically inhibits HSV-1 production but not HSV-2 production (Langeland et al., Biochem Biophys. Res. Commun. 141:198-203, 1986). We now show that neomycin had no effect on cellular protein synthesis, as judged by the appearance of 35S-labeled polypeptides separated by polyacrylamide gel electrophoresis. Virus-induced polypeptides, however, were strongly inhibited at neomycin concentrations above 2 mM. Comparison among different aminoglycosides showed a variation in inhibition of HSV-1 production that paralleled the cationic charge of the aminoglycosides. HSV-1 receptor binding at 4 degrees C was completely inhibited by neomycin. At 37 degrees C both receptor binding and internalization, as measured by an indirect assay, appeared to be inhibited by more than 90%. The effect of neomycin on the infection was almost immediate upon the addition of the drug and preceded virus internalization. Possible mechanisms of the neomycin effect are discussed.     

Intracellular interleukin-1 receptor antagonist type 1 antagonizes the stimulatory effect of interleukin-1 alpha precursor on cell motility

Interleukin (IL)-1alpha, a proinflammatory cytokine, is produced as a 33 kDa protein precursor (preIL-1alpha) which is cleaved to generate the 17 kDa C-terminal mature IL-1alpha (mIL-1alpha) and the 16kDa N-terminal IL-1alpha propiece (NIL-1alpha). The biological effect of IL-1alpha is regulated by the IL-1 receptor antagonist (IL-1Ra), its naturally occurring inhibitor. Four different isoforms of the IL-1Ra have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Whether the icIL-1Ra1 isoform can antagonize some of the biological effects of intracellular IL-1alpha is still unknown. The aim of this study is to investigate effects of preIL-1alpha and icIL-1Ra1 on cell motility in stably transfected ECV304 cells. We show that expression of preIL-1alpha in ECV304 cells significantly increases cell motility. Furthermore, transfection with NIL-1alpha propiece also increases cell motility whereas this stimulatory effect was not observed by addition of exogenous mIL-1alpha, suggesting an intracellular effect of preIL-1alpha mediated by NIL-1alpha propiece. Co-transfection of ECV304 cells with icIL-1Ra1 completely antagonizes the stimulatory effect of preIL-1alpha and NIL-1alpha propiece on cell motility. In conclusion, NIL-1alpha propiece increases ECV304 cell motility and icIL-1Ra1 exerts intracellular functions regulating this stimulatory effect.     

Physiologic role of interleukin-1 receptor antagonist

Recent studies have described the spontaneous development of arthritis or vasculitis in IL-1 receptor antagonist (IL-1Ra) knockout mice bred on specific and different genetic backgrounds. The levels of both secreted and intracellular isoforms of IL-1Ra produced in the rheumatoid joint or in the arterial wall may not be adequate to effectively inhibit the excess amounts of locally produced IL-1. Thus, an imbalance between IL-1 and IL-1Ra may predispose to local inflammatory disease in particular tissues in the presence of other as yet unknown genetically influenced factors.     

Intracellular IL-1 receptor antagonist type 1 inhibits IL-1-induced cytokine production in keratinocytes through binding to the third component of the COP9 signalosome

The IL-1 receptor antagonist (IL-1Ra) exists in four isoforms, three of which lack signal peptides and are primarily intracellular proteins. The biologic roles of the intracellular isoforms of IL-1Ra have remained unknown. The objective of these studies was to determine whether the major intracellular isoform of IL-1Ra 18-kDa type 1 (icIL-1Ra1), mediated unique functions inside cells. A yeast two-hybrid screen with HeLa cell lysates revealed specific binding of icIL-1Ra1, and not of the other IL-1Ra isoforms, to the third component of the COP9 signalosome complex (CSN3). This binding was confirmed by Far Western blot analysis, sedimentation on a glycerol gradient, glutathione pull-down experiments, and coimmunoprecipitation. In addition to binding specifically to CSN3, icIL-1Ra1 inhibited phosphorylation of p53, c-Jun, and IkappaB by the crude CSN-associated kinase and of p53 by recombinant protein kinase CK2 and protein kinase D, both associated with CSN3. The biologic relevance of the interaction between icIL-1Ra1 and CSN3 was demonstrated in the keratinocyte cell lines KB and A431, both possessing abundant CSN3. A431 cells exhibited high levels of icIL-1Ra1 but lacked both detectable IL-1alpha-induced IL-6 and IL-8 production and phosphorylation of p38 MAPK. KB cells displayed the opposite pattern which was reversed after transfection with icIL-1Ra1 mRNA. Inhibition of CSN3 or of icIL-1Ra1 production through gene knockdown with specific small interfering RNA in A431 cells each led to an inhibition of IL-1alpha-induced IL-6 and IL-8 production. Thus, icIL-1Ra1 exhibits unique anti-inflammatory properties inside cells through binding to CSN3 with subsequent inhibition of the p38 MAPK signal transduction pathway.     

重组人IL-1ra在大肠杆菌中的可溶性表达

利用PCR技术从含有IL-1ra的质粒上扩增IL-1ra基因,经过序列测定后插入表达载体pTIG-Trx,并转化大肠杆菌BL21(DE3),用IPTG进行诱导表达。经SDS-PAGE分析显示,IL-1ra表达质粒在大肠杆菌中的诱导表达产物出现相对分子量大约为17000的一条新生蛋白质带,其大小与预期结果一致,经Western和ELISA分析,证明该带即为目的蛋白,SDS-PAGE显示目的蛋白全部以可溶性蛋白的形式存在。超声破碎后,上清经金属螯和层析纯化获得纯度约为98％的蛋白样品。     

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.     

Human recombinant interleukin-1 receptor antagonist inhibits lymphocyte blastogenesis induced by concanavalin A. Restorative effect of hrIL-1.

Interleukin-1 (IL-1), mainly produced by monocyte-macrophages, is a polypeptide cytokine with pleiotropic biological effects. IL-1 plays an important role in mediating immune response and inflammation. Recently a natural inhibitor to IL-1 has been discovered, interleukin-1 receptor antagonist (IL-1ra), produced by human monocytes cultured on adherent IgG which binds to the IL-1 receptors. In our study we found that the pretreatment of cells with serial dilutions of IL-1ra (250 ng/ml-2.5 pg/ml) inhibits, in a dose-dependent manner, lymphocyte DNA synthesis stimulated with Con A (10 micrograms/ml). IL-1ra did not have any effect on resting peripheral blood mononuclear cells (PBMC). Time course experiments show that IL-1ra at 250 ng/ml has its maximum inhibitory effect on lymphocyte blastogenesis when cells are pretreated 2 h before Con A. No effect was found when hrIL-1ra was added after Con A. Moreover, hrIL-1ra also inhibits the enhancing effects of exogenous hrIL-1 (400, 200, 100 and 50 ng/ml) on lymphocytes stimulated with Con A; while when hrIL-1ra was used on cells treated with only Con A, the inhibition was more pronounced. When PBMC were removed from monocytes, by adherence, the Con A-treated lymphocytes were not influenced by 2 h pretreatment of hrIL-1ra; while a strong inhibition was found when exogenous hrIL-1 was added at different concentrations. In addition, hrIL-1ra also inhibits the enhancing effect of hrIL-2 on lymphocyte DNA synthesis. In another set of experiments PBMC were pretreated with hrIL-1ra (250 ng/ml) for 2 h and then added LPs (10 ng/ml) and IL-1 alpha generation was determined using ELISA. In these experiments IL-1ra completely abolished the generation of IL-1 alpha. These data suggest that hrIL-1ra exhibits a dose-response inhibition of lymphocyte blastogenesis induced by Con A, probably through the down-regulation of IL-1 synthesis necessary as an early signal for T-cell activation and IL-2 production.     

Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3) increased concentrations of sgp130 should produce an efficient buffering effect on the IL-6/sIL-6R complex without incidence on the level of free IL-6. According to this model, the IL-6/sIL-6R complex appears to be an important support of IL-6 signaling in the most commonly encountered in vivo situations. The concentration of this complex is directly under the control of the concentration of sIL-6R; its bio-availability should be efficiently buffered by increased sgp130 concentrations.     

Nuclear internalisation and DNA binding activities of interleukin-1, interleukin-1 receptor and interleukin-1/receptor complexes.

This paper presents evidence to suggest that interleukin-1 alpha as a complex with its receptor is able to express DNA binding activity. Both the interleukin-1/receptor complex and the interleukin-1 receptor appear to be able to bind to DNA, however interleukin-1 on its own showed no binding activity. Interleukin-1 was found to be internalised into the nuclei of all cells examined (EL4, MEL, HL-60, K562, THP-1 and Jurkat cells). The data suggest the possible modulation of genes by interaction of interleukin-1/receptor complexes with DNA structures.