The structure of the interleukin-15 alpha receptor and its implications for ligand binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.     

Elucidation of the interleukin-15 binding site on its alpha receptor by NMR

The cytokine interleukin-15 (IL-15) signals through the formation of a quaternary receptor complex composed of an IL-15-specific alpha receptor, together with beta and gammac receptors that are shared with interleukin-2 (IL-2). The initiating step in the formation of this signaling complex is the interaction between IL-15 and IL-15Ralpha, which is a single sushi domain bearing strong structural homology to one of the two sushi domains of IL-2Ralpha. The crystal structure of the IL2-Ralpha/IL-2 complex has been determined, however little is known about the analogous IL-15Ralpha/IL-15 binding interaction. Here we show that recombinant IL-15 can be overexpressed as a stable complex in the presence of its high affinity receptor, IL-15Ralpha. We find that this complex is 10-fold more active than IL-15 alone in stimulating proliferation and survival of memory phenotype CD8 T cells. To probe the ligand/receptor interface, we used solution NMR to map chemical shifts on 15N-labeled IL-15Ralpha in complex with unlabeled IL-15. Our results predict that the binding surface on IL-15Ralpha involves strands C and D, similar to IL-2Ralpha. The interface, as predicted here, leaves open the possibility of trans-presentation of IL-15 by IL-15Ralpha on an opposing cell.     

Nuclear and mitochondrial interaction involving mt-Nd2 leads to increased mitochondrial reactive oxygen species production

NADH dehydrogenase subunit 2, encoded by the mtDNA, has been associated with resistance to autoimmune type I diabetes (T1D) in a case control study. Recently, we confirmed a role for the mouse ortholog of the protective allele (mt-Nd2(a)) in resistance to T1D using genetic analysis of outcrosses between T1D-resistant ALR and T1D-susceptible NOD mice. We sought to determine the mechanism of disease protection by elucidating whether mt-Nd2(a) affects basal mitochondrial function or mitochondrial function in the presence of oxidative stress. Two lines of reciprocal conplastic mouse strains were generated: one with ALR nuclear DNA and NOD mtDNA (ALR.mt(NOD)) and the reciprocal with NOD nuclear DNA and ALR mtDNA (NOD.mt(ALR)). Basal mitochondrial respiration, transmembrane potential, and electron transport system enzymatic activities showed no difference among the strains. However, ALR.mt(NOD) mitochondria supported by either complex I or complex II substrates produced significantly more reactive oxygen species when compared with both parental strains, NOD.mt(ALR) or C57BL/6 controls. Nitric oxide inhibited respiration to a similar extent for mitochondria from the five strains due to competitive antagonism with molecular oxygen at complex IV. Superoxide and hydrogen peroxide generated by xanthine oxidase did not significantly decrease complex I function. The protein nitrating agents peroxynitrite or nitrogen dioxide radicals significantly decreased complex I function but with no significant difference among the five strains. In summary, mt-Nd2(a) does not confer elevated resistance to oxidative stress; however, it plays a critical role in the control of the mitochondrial reactive oxygen species production.     

Crystal Structure of the interleukin-15.interleukin-15 receptor alpha complex: insights into trans and cis presentation

Interleukin (IL)-15 is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is unique among cytokines due to its participation in a trans signaling mechanism in which IL-15 receptor alpha (IL-15Ralpha) from one subset of cells presents IL-15 to neighboring IL-2Rbeta/gammac-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15Ralpha. The structure reveals that the alpha receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions, permits specific interactions with IL-15Ralpha that account for the exceptionally high affinity of the IL-15.IL-15Ralpha complex. Interestingly, analysis of the topology of IL-15 and IL-15Ralpha at the IL-15.IL-15Ralpha interface suggests that IL-15 should be capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that IL-15 is capable of efficiently signaling in cis through IL-15Ralpha and IL-2Rbeta/gammac expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15Ralpha permits IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15.IL-15Ralpha.IL-2Rbeta.gammac quaternary complex assembly through the use of molecular modeling.     

How a cytokine is chaperoned through the secretory pathway by complexing with its own receptor: lessons from interleukin-15 (IL-15)/IL-15 receptor alpha

While it is well appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is known about additional roles for cytokine receptor components in the control of ligand transport and secretion. Here, we show that interleukin-15 (IL-15) translocation into the endoplasmic reticulum occurs independently of the presence of IL-15 receptor α (IL-15Rα). Subsequently, however, IL-15 is transported through the Golgi apparatus only in association with IL-15Rα and then is secreted. This intracellular IL-15/IL-15Rα complex already is formed in the endoplasmic reticulum and, thus, enables the further trafficking of complexed IL-15 through the secretory pathway. Just transfecting IL-15Rα in cells, which transcribe but normally do not secrete IL-15, suffices to induce IL-15 secretion. Thus, we provide the first evidence of how a cytokine is chaperoned through the secretory pathway by complexing with its own high-affinity receptor and show that IL-15/IL-15Rα offers an excellent model system for the further exploration of this novel mechanism for the control of cytokine secretion.     

Intracellular interactions of transferrin and its receptor during biosynthesis

The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during biosynthesis were studied in the human hepatoma cell line HepG2, which synthesizes both. Early during biosynthesis the Tfr monomer is converted to a disulfide-linked Tfr dimer. The Tfr monomer is not able to bind Tf, but Tf binding is observed as soon as the covalent Tfr dimer is formed and can take place in the ER. The Tf-Tfr complex is transported through the Golgi reticulum and trans-Golgi reticulum (TGR) and is ultimately delivered to an acidic compartment, where Tf releases its Fe3+. We did not observe conversion of Tf to apoTf in the TGR, showing that the part of the TGR passed by secreted Tf has a pH higher than 5.5. We conclude that when a ligand-receptor combination is synthesized by one and the same cell, ligand and receptor can interact during biosynthesis and be transported to the cell surface.     

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.     

Association of interleukin-15 protein and interleukin-15 receptor genetic variation with resistance exercise training responses.

Interleukin-15 (IL-15) is an anabolic cytokine that is produced in skeletal muscle and directly affects muscle anabolism in animal and in vitro models. The contribution of IL-15 variability in muscle responses to 10 wk of resistance exercise training in young men and women was examined by measuring acute and chronic changes in IL-15 protein in plasma and characterizing genetic variation in the IL-15 receptor-alpha gene (IL15RA). Participants trained 3 days a week at 75% of one repetition maximum, performing three sets (6-10 repetitions) of 13 resistance exercises. Plasma IL-15 protein was significantly increased (P < 0.05) immediately after acute resistance exercise but did not change with training and was not associated with variability in muscle responses with training. A single nucleotide polymorphism in exon 7 of IL15RA was strongly associated with muscle hypertrophy and accounted for 7.1% of the variation in regression modeling. A polymorphism in exon 4 was also independently associated with muscle hypertrophy and accounted for an additional 3.5% of the variation in hypertrophy. These results suggest that IL-15 is an important mediator of muscle mass response to resistance exercise training in humans and that genetic variation in IL15RA accounts for a significant proportion of the variability in this response.     

Stress-induced phosphorylation of PACT reduces its interaction with TRBP and leads to PKR activation

PACT is a stress-modulated activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) and is an important regulator of PKR-dependent signaling pathways. Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation by PACT leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. In addition to positive regulation by PACT, PKR activity in cells is also negatively regulated by TRBP. In this study, we demonstrate for the first time that stress-induced phosphorylation at serine 287 significantly increases PACT's ability to activate PKR by weakening PACT's interaction with TRBP. A non-phosphorylatable alanine substitution mutant at this position causes enhanced interaction of PACT with TRBP and leads to a loss of PKR activation. Furthermore, TRBP overexpression in cells is unable to block apoptosis induced by a phospho-mimetic, constitutively active PACT mutant. These results demonstrate for the first time that stress-induced PACT phosphorylation functions to free PACT from the inhibitory interaction with TRBP and also to enhance its interaction with PKR.     

Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells

Geobacter sulfurreducens developed highly structured, multilayer biofilms on the anode surface of a microbial fuel cell converting acetate to electricity. Cells at a distance from the anode remained viable, and there was no decrease in the efficiency of current production as the thickness of the biofilm increased. Genetic studies demonstrated that efficient electron transfer through the biofilm required the presence of electrically conductive pili. These pili may represent an electronic network permeating the biofilm that can promote long-range electrical transfer in an energy-efficient manner, increasing electricity production more than 10-fold.     

Soluble interleukin-15 receptor alpha (IL-15R alpha)-sushi as a selective and potent agonist of IL-15 action through IL-15R beta/gamma. Hyperagonist IL-15 x IL-15R alpha fusion proteins

Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15R alpha, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.     

Effect of soluble interleukin-6 receptor alpha and interleukin-6 secreted by polymorphonuclear leukocytes on tumor necrosis factor-alpha expression and its production by peripheral blood mononuclear cells

BACKGROUND: It has recently been shown that soluble interleukin-6 receptor (sIL-6R) alone or complexed with interleukin (IL)-6, besides their regulatory role in a wide variety of both normal and abnormal biologic reactions mediated by IL-6, could be an effective stimulator of the cell function. AIMS: The key question of the present study is whether the sIL-6Ralpha or sIL-6R with IL-6 released by polymorphonuclear leukocytes (PMN) can influence cytokine secretion such as tumor necrosis factor-alpha (TNF-alpha) by peripheral blood mononuclear cells (PBMC), which together with PMN develop the inflammatory and immune response of a host. METHODS: Cells were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 1 h at 37 degrees C in 5% CO(2). After incubation, the culture supernatant of PMN was removed and was added to PBMC. The PBMC were cultured for 1 h at 37 degrees C in the same conditions. In the culture supernatants and lysates of PMN, we examined the concentrations of sIL-6R by enzyme-linked immunosorbent assay (ELISA). TNF-alpha was measured at both protein and mRNA levels. Protein levels were determined by ELISA. To examine TNF-alpha mRNA expression, we isolated mRNA from PBMC after culture, using TRIZOL Reagent. The quantity of mRNA TNF-alpha was determined by the Quantikine mRNA assay. RESULTS AND CONCLUSION: The results obtained revealed that sIL-6R with IL-6 secreted by PMN may play a regulatory role in the immune response by modulating the TNF-alpha expression and its production by PBMC. This may have a significant influence on an early phase of the inflammation and other reactions mediated by TNF-alpha.     

Parathyroid hormone induces hepatic production of bioactive interleukin-6 and its soluble receptor

Interleukin-6 (IL-6) is an important mediator of parathyroid hormone (PTH)-induced bone resorption. Serum levels of IL-6 and its soluble receptor (IL-6sR) are regulated in part by PTH. The PTH/PTH-related protein type 1 receptor is highly expressed in the liver, and in the current study we investigated whether the liver produces IL-6 or IL-6sR in response to PTH. Perfusion of the isolated rat liver with PTH-(1-84) stimulated rapid, dose-dependent production of bioactive IL-6 and the IL-6sR. These effects were observed at near physiological concentrations of the hormone such that 1 pM PTH induced hepatic IL-6 production at a rate of approximately 0.6 ng/min. In vitro, hepatocytes, hepatic endothelial cells, and Kupffer cells, but not hepatic stellate cells, were each found to produce both IL-6 and IL-6sR in response to higher (10 nM) concentrations of PTH. Our data suggest that hepatic-derived IL-6 and IL-6sR contribute to the increase in circulating levels of these cytokines induced by PTH in vivo and raise the possibility that PTH-induced, liver-derived IL-6 may exert endocrine effects on tissues such as bone.     

Molecular analysis of the interaction of LCMV with its cellular receptor [alpha]-dystroglycan

alpha-Dystroglycan (DG) has been identified as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). This subunit of DG is a highly versatile cell surface molecule that provides a molecular link between the extracellular matrix (ECM) and a beta-DG transmembrane component, which interacts with the actin-based cytoskeleton. In addition, DG exhibits a complex pattern of interaction with a wide variety of ECM and cellular proteins. In the present study, we characterized the binding of LCMV to alpha-DG and addressed the role of alpha-DG-associated host-derived proteins in virus infection. We found that the COOH-terminal region of alpha-DG's first globular domain and the NH2-terminal region of the mucin-related structures of alpha-DG together form the binding site for LCMV. The virus-alpha-DG binding unlike ECM alpha-DG interactions was not dependent on divalent cations. Despite such differences in binding, LCMV and laminin-1 use, in part, an overlapping binding site on alpha-DG, and the ability of an LCMV isolate to compete with laminin-1 for receptor binding is determined by its binding affinity to alpha-DG. This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to alpha-DG, tissue tropism, and pathological potential. LCMV strains and variants with high binding affinity to alpha-DG but not low affinity binders are able to infect CD11c+ dendritic cells, which express alpha-DG at their surface. Infection followed by dysfunction of these antigen-presenting cells contributes to immunosuppression and persistent viral infection in vivo.     

The human IgA-Fc alpha receptor interaction and its blockade by streptococcal IgA-binding proteins

IgA plays a key role in immune defence of the mucosal surfaces. IgA can trigger elimination mechanisms against pathogens through the interaction of its Fc region with Fc alpha Rs (receptors specific for the Fc region of IgA) present on neutrophils, macrophages, monocytes and eosinophils. The human Fc alpha R (CD89) shares homology with receptors specific for the Fc region of IgG (Fc gamma Rs) and IgE (Fc epsilon RIs), but is a more distantly related member of the receptor family. CD89 interacts with residues lying at the interface of the two domains of IgA Fc, a site quite distinct from the homologous regions at the top of IgG and IgE Fc recognized by Fc gamma R and Fc epsilon RI respectively. Certain pathogenic bacteria express surface proteins that bind to human IgA Fc. Experiments with domain-swap antibodies and mutant IgAs indicate that binding of three such proteins (Sir22 and Arp4 of Streptococcus pyogenes and beta protein of group B streptococci) depend on sites in the Fc interdomain region of IgA, the binding region also used by CD89. Further, we have found that the streptococcal proteins can inhibit interaction of IgA with CD89, and have thereby identified a mechanism by which a bacterial IgA-binding protein may modulate IgA effector function.     

Interferon receptor interaction. Internalization of interferon alpha 2 and modulation of its receptor on human cells

Studies reported earlier [ Joshi et al. (1982) J. Biol. Chem. 257, 13884-13887] have indicated that human interferon-alpha 2 (HuIFN-alpha 2) binds to a specific macromolecular receptor on human cells as identified by cross-linking with bifunctional cross-linking reagents and analysis by polyacrylamide gel electrophoresis. We have carried out experiments to investigate the fate of the interferon-receptor complex on the cell surface under conditions which lead to cellular response. As analyzed by cross-linking and gel electrophoresis, the interferon-receptor complex, formed on incubation with 125I-IFN-alpha 2 at 4 degrees C, persisted at the cell surface for several hours at 4 degrees C; however, if the cells were switched to 37 degrees C, there was a rapid decline in the complex, apparently due to a loss of the interferon receptors from the cell surface. This was associated with an internalization of the 125I-interferon as indicated by the fact that, on incubation at 37 degrees C, an appreciable fraction of the cell-associated interferon (approximately equal to 50%) became resistant to trypsin digestion, or dissociation on incubation in growth medium or low-pH buffer. A large fraction of the trypsin-resistant (internalized) 125I-labeled material migrated as intact interferon in polyacrylamide gels, and it was immunoprecipitated by anti-(HuIFN-alpha)antibodies but not by anti-(HuIFN-beta)antibodies. The bulk of the internalized 125I-interferon was recovered in a particulate fraction and, on cross-linking with disuccinimidyl suberate, a 150000-Mr complex could be detected. The results suggest that interferon may be internalized as a complex with the receptor, which may account for the loss of the interferon-receptors on the cell surface. This modulation of the IFN-alpha/beta receptors was induced by HuIFN-alpha and HuIFN-beta but not by HuIFN-gamma. The recovery of the IFN-alpha/beta receptors, lost upon incubation with HuIFN-alpha, took several hours and required protein synthesis. The significance of the results is discussed.     

Epstein-barr virus immortalization of human B-cells leads to stabilization of hypoxia-induced factor 1 alpha, congruent with the warburg effect

Background

Epstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied.

Methods and Findings

Using Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate.

Conclusions

Our data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.