The roles of interleukin-1 receptor accessory protein in certain inflammatory conditions.

Interleukin-1 receptor accessory protein (IL-1RAcP) is a member of the immunoglobulin superfamily proteins consisting of soluble and membranous isoforms. IL-1RAcP plays an essential role in the signaling of the IL-1 family cytokines such as IL-1, IL-33 and IL-36, as well as tyrosine kinases FLT3 and C-Kit. IL-1RAcP generally initiates inflammatory signaling pathway through the recruitment of signaling mediators, including MYD88 and IRAK. Chronic inflammation following prolonged signaling of cytokine receptors is a critical process in the pathogenesis of many inflammatory disorders, including autoimmunity, obesity, psoriasis, type 1 diabetes, endometriosis, preeclampsia and Alzheimer's disease. Recently IL-1RAcP aberrant signaling has been considered to play a central role in the pathogenesis of these chronic inflammatory diseases. Targeting IL-1RAcP signaling pathway that was recently considered in clinical trials related to malignancies also indicates its potential as therapeutic target for the inflammatory and autoimmune diseases. This review summarizes the molecular structure, components associated with IL-1RAcP signaling pathways, and their involvement in the pathogenesis of different inflammatory diseases. We will also discuss the effect of IL-1RAcP inhibition for treatment proposes.

Crystal structure of the Toll/interleukin-1 receptor (TIR) domain of IL-1R10 provides structural insights into TIR domain signalling.

The Toll/interleukin-1 receptor (TIR) domains are key innate immune signalling modules. Here, we present the crystal structure of the TIR domain of human interleukin-1 receptor 10 (IL-1R10), also called interleukin 1 receptor accessory protein like 2. It is similar to that of IL-1R9 (IL-1RAPL1) but shows significant structural differences to those from Toll-like receptors (TLRs) and the adaptor proteins MyD88 adaptor-like protein (MAL) and MyD88. Interactions of TIR domains in their respective crystals and the higher-order assemblies (MAL and MyD88) reveal the presence of a common 'BCD surface', suggesting its functional significance. We also show that the TIR domains of IL-1R10 and IL-1R9 lack NADase activity, consistent with their structures. Our study provides a foundation for unravelling the functions of IL-1R9 and IL-1R10.

Glycan-mediated functional assembly of IL-1RI: structural insights into completion of the current description for immune response.

Interleukin 1 Receptor type I (IL-1RI) is a multi-domain transmembrane receptor that triggers the inflammatory response. Understanding its detailed mechanism of action is crucial for treating immune disorders. IL-1RI is activated upon formation of its functional assembly that occurs by binding of the IL-1 cytokine and the accessory protein (Il-1RAcP) to it. X-ray crystallography, small-Angle X-ray Scattering and molecular dynamics simulation studies showed that IL-1RI adopts two types of 'compact' and 'extended' conformational states in its dynamical pattern. Furthermore, glycosylation has shown to play a critical role in its activation process. Here, classical and accelerated atomistic molecular dynamics were carried out to examine the role of full glycosylation of IL-1RI and IL-1RAcP in arrangement of the functional assembly. Simulations showed that the 'compact' and 'extended' IL-1RI form two types of 'cytokine-inaccessible-non-signaling' and 'cytokine-accessible-signaling' assemblies with the IL-1RacP, respectively that are both abiding in the presence of glycans. Suggesting that the cytokine binding to IL-1RI is not required for the formation of IL-1RI-IL-1RAcP complex and the 'compact' complex could act as a down-regulatory mechanism. The 'extended' complex is maintained by formation of several persistent hydrogen bonds between the IL-1RI-IL-1RAcP inter-connected glycans. Taken together, it was shown that full glycosylation regulates formation of the IL-1RI functional assembly and play critical role in cytokine biding and triggering the IL-1RI involved downstream pathways in the cell.Communicated by Ramaswamy H. Sarma.

Structure-based Development of Human Interleukin-1ß-Specific Antibody That Simultaneously Inhibits Binding to Both IL-1RI and IL-1RAcP.

Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.

Structural and Functional Attributes of the Interleukin-36 Receptor.

Signal transduction by the IL-36 receptor (IL-36R) is linked to several human diseases. However, the structure and function of the IL-36R is not well understood. A molecular model of the IL-36R complex was generated and a cell-based reporter assay was established to assess the signal transduction of recombinant subunits of the IL-36R. Mutational analyses and functional assays have identified residues of the receptor subunit IL-1Rrp2 needed for cytokine recognition, stable protein expression, disulfide bond formation and glycosylation that are critical for signal transduction. We also observed that, overexpression of ectodomain (ECD) of Il-1Rrp2 or IL-1RAcP exhibited dominant-negative effect on IL-36R signaling. The presence of IL-36 cytokine significantly increased the interaction of IL-1Rrp2 ECD with the co-receptor IL-1RAcP. Finally, we found that single nucleotide polymorphism A471T in the Toll-interleukin 1 receptor domain (TIR) of the IL-1Rrp2 that is present in ∼2% of the human population, down-regulated IL-36R signaling by a decrease of interaction with IL-1RAcP.

Novel IL1RAPL1 mutations associated with intellectual disability impair synaptogenesis.

Mutations in interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene have been associated with non-syndromic intellectual disability (ID) and autism spectrum disorder. This protein interacts with synaptic partners like PSD-95 and PTPδ, regulating the formation and function of excitatory synapses. The aim of this work was to characterize the synaptic consequences of three IL1RAPL1 mutations, two novel causing the deletion of exon 6 (Δex6) and one point mutation (C31R), identified in patients with ID. Using immunofluorescence and electrophysiological recordings, we examined the effects of IL1RAPL1 mutant over-expression on synapse formation and function in cultured rodent hippocampal neurons. Δex6 but not C31R mutation leads to IL1RAPL1 protein instability and mislocalization within dendrites. Analysis of different markers of excitatory synapses and sEPSC recording revealed that both mutants fail to induce pre- and post-synaptic differentiation, contrary to WT IL1RAPL1 protein. Cell aggregation and immunoprecipitation assays in HEK293 cells showed a reduction of the interaction between IL1RAPL1 mutants and PTPδ that could explain the observed synaptogenic defect in neurons. However, these mutants do not affect all cellular signaling because their over-expression still activates JNK pathway. We conclude that both mutations described in this study lead to a partial loss of function of the IL1RAPL1 protein through different mechanisms. Our work highlights the important function of the trans-synaptic PTPδ/IL1RAPL1 interaction in synaptogenesis and as such in ID in the patients.

The structural basis for receptor recognition of human interleukin-18.

Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and ß (Rß) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors' recognition mode for IL-18 is similar to IL-1ß; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity.

Blood soluble interleukin 1 receptor accessory protein levels are consistently low throughout the menstrual cycle of women with endometriosis.

BACKGROUND: A deficiency in the counter-regulatory mechanisms of interleukin 1 (IL1) may play a significant role in endometriosis pathogenesis and associated chronic inflammation. The aim of this study was to investigate peripheral blood levels of soluble IL1 receptor accessory protein (sIL1RAP), a potent natural inhibitor of IL1, in women with and without endometriosis. METHODS: Peripheral blood samples were collected from women with endometriosis (n = 47) consulting for infertility, pelvic pain or tubal ligation, in whom the disease was diagnosed at laparoscopy. Control healthy women (n = 27) were requesting tubal ligation or reanastomosis and had no visible evidence of endometriosis at laparoscopy. sIL1RAP levels were determined by ELISA, whereas estradiol (E2) and progesterone (P4) levels were determined by competitive immunoassays. RESULTS: sIL1RAP levels were significantly decreased in women with early endometriosis stages compared to controls (p < 0.05) and markedly during the proliferative phase of the menstrual cycle (p < 0.001). Actually, while sIL1RAP were significantly increased in the proliferative compared to the secretory phase in normal women (p < 0.0001) and peaked at the end of this phase, sIL1RAP remained consistently low and showed non-significant variations throughout the menstrual cycle in women with endometriosis. CONCLUSIONS: Lower circulating levels of sIL1RAP points to a significant impairment in the counter-regulatory mechanisms of IL1, which in view of the cytokine's potent inflammatory and growth-promoting properties may play a significant role in the pathophysiology of endometriosis.

The structural pathway of interleukin 1 (IL-1) initiated signaling reveals mechanisms of oncogenic mutations and SNPs in inflammation and cancer.

Interleukin-1 (IL-1) is a large cytokine family closely related to innate immunity and inflammation. IL-1 proteins are key players in signaling pathways such as apoptosis, TLR, MAPK, NLR and NF-κB. The IL-1 pathway is also associated with cancer, and chronic inflammation increases the risk of tumor development via oncogenic mutations. Here we illustrate that the structures of interfaces between proteins in this pathway bearing the mutations may reveal how. Proteins are frequently regulated via their interactions, which can turn them ON or OFF. We show that oncogenic mutations are significantly at or adjoining interface regions, and can abolish (or enhance) the protein-protein interaction, making the protein constitutively active (or inactive, if it is a repressor). We combine known structures of protein-protein complexes and those that we have predicted for the IL-1 pathway, and integrate them with literature information. In the reconstructed pathway there are 104 interactions between proteins whose three dimensional structures are experimentally identified; only 15 have experimentally-determined structures of the interacting complexes. By predicting the protein-protein complexes throughout the pathway via the PRISM algorithm, the structural coverage increases from 15% to 71%. In silico mutagenesis and comparison of the predicted binding energies reveal the mechanisms of how oncogenic and single nucleotide polymorphism (SNP) mutations can abrogate the interactions or increase the binding affinity of the mutant to the native partner. Computational mapping of mutations on the interface of the predicted complexes may constitute a powerful strategy to explain the mechanisms of activation/inhibition. It can also help explain how an oncogenic mutation or SNP works.

Structural insights into the interaction of IL-33 with its receptors.

Interleukin (IL)-33 is an important member of the IL-1 family that has pleiotropic activities in innate and adaptive immune responses in host defense and disease. It signals through its ligand-binding primary receptor ST2 and IL-1 receptor accessory protein (IL-1RAcP), both of which are members of the IL-1 receptor family. To clarify the interaction of IL-33 with its receptors, we determined the crystal structure of IL-33 in complex with the ectodomain of ST2 at a resolution of 3.27 Å. Coupled with structure-based mutagenesis and binding assay, the structural results define the molecular mechanism by which ST2 specifically recognizes IL-33. Structural comparison with other ligand-receptor complexes in the IL-1 family indicates that surface-charge complementarity is critical in determining ligand-binding specificity of IL-1 primary receptors. Combined crystallography and small-angle X-ray-scattering studies reveal that ST2 possesses hinge flexibility between the D3 domain and D1D2 module, whereas IL-1RAcP exhibits a rigid conformation in the unbound state in solution. The molecular flexibility of ST2 provides structural insights into domain-level conformational change of IL-1 primary receptors upon ligand binding, and the rigidity of IL-1RAcP explains its inability to bind ligands directly. The solution architecture of IL-33-ST2-IL-1RAcP complex from small-angle X-ray-scattering analysis resembles IL-1ß-IL-1RII-IL-1RAcP and IL-1ß-IL-1RI-IL-1RAcP crystal structures. The collective results confer IL-33 structure-function relationships, supporting and extending a general model for ligand-receptor assembly and activation in the IL-1 family.

IL1RAPL1 associated with mental retardation and autism regulates the formation and stabilization of glutamatergic synapses of cortical neurons through RhoA signaling pathway.

Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) is associated with X-linked mental retardation and autism spectrum disorder. We found that IL1RAPL1 regulates synapse formation of cortical neurons. To investigate how IL1RAPL1 controls synapse formation, we here screened IL1RAPL1-interacting proteins by affinity chromatography and mass spectroscopy. IL1RAPL1 interacted with Mcf2-like (Mcf2l), a Rho guanine nucleotide exchange factor, through the cytoplasmic Toll/IL-1 receptor domain. Knockdown of endogenous Mcf2l and treatment with an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of RhoA, suppressed IL1RAPL1-induced excitatory synapse formation of cortical neurons. Furthermore, we found that the expression of IL1RAPL1 affected the turnover of AMPA receptor subunits. Insertion of GluA1-containing AMPA receptors to the cell surface was decreased, whereas that of AMPA receptors composed of GluA2/3 was enhanced. Mcf2l knockdown and ROCK inhibitor treatment diminished the IL1RAPL1-induced changes of AMPA receptor subunit insertions. Our results suggest that Mcf2l-RhoA-ROCK signaling pathway mediates IL1RAPL1-dependent formation and stabilization of glutamatergic synapses of cortical neurons.

One target-two different binding modes: structural insights into gevokizumab and canakinumab interactions to interleukin-1ß.

Interleukin-1ß (IL-1ß) is a key orchestrator in inflammatory and several immune responses. IL-1ß exerts its effects through interleukin-1 receptor type I (IL-1RI) and interleukin-1 receptor accessory protein (IL-1RAcP), which together form a heterotrimeric signaling-competent complex. Canakinumab and gevokizumab are highly specific IL-1ß monoclonal antibodies. Canakinumab is known to neutralize IL-1ß by competing for binding to IL-1R and therefore blocking signaling by the antigen:antibody complex. Gevokizumab is claimed to be a regulatory therapeutic antibody that modulates IL-1ß bioactivity by reducing the affinity for its IL-1RI:IL-1RAcP signaling complex. How IL-1ß signaling is affected by both canakinumab and gevokizumab was not yet experimentally determined. We have analyzed the crystal structures of canakinumab and gevokizumab antibody binding fragment (Fab) as well as of their binary complexes with IL-1ß. Furthermore, we characterized the epitopes on IL-1ß employed by the antibodies by NMR epitope mapping studies. The direct comparison of NMR and X-ray data shows that the epitope defined by the crystal structure encompasses predominantly those residues whose NMR resonances are severely perturbed upon complex formation. The antigen:Fab co-structures confirm the previously identified key contact residues on IL-1ß and provide insight into the mechanisms leading to their distinct modulation of IL-1ß signaling. A significant steric overlap of the binding interfaces of IL-1R and canakinumab on IL-1ß causes competitive inhibition of the association of IL-1ß and its receptor. In contrast, gevokizumab occupies an allosteric site on IL-1ß and complex formation results in a minor reduction of binding affinity to IL-1RI. This suggests two different mechanisms of IL-1ß pathway attenuation.

Structure of the activating IL-1 receptor signaling complex.

Interleukin-1 (IL-1)-family cytokines are mediators of innate and adaptive immunity. They exert proinflammatory effects by binding a primary receptor that recruits a receptor accessory protein to form a signaling-competent heterotrimeric complex. Here we present the crystal structure of IL-1ß bound to its primary receptor IL-1RI and its receptor accessory protein IL-1RAcP, providing insight into how IL-1-type cytokines initiate signaling and revealing an evolutionary relationship with the fibroblast growth factor receptor family.

The X-linked intellectual disability protein IL1RAPL1 regulates excitatory synapse formation by binding PTPδ and RhoGAP2.

Mutations of the Interleukin-1-receptor accessory protein like 1 (IL1RAPL1) gene are associated with cognitive impairment ranging from non-syndromic X-linked mental retardation to autism. IL1RAPL1 belongs to a novel family of IL1/Toll receptors, which is localized at excitatory synapses and interacts with PSD-95. We previously showed that IL1RAPL1 regulates the synaptic localization of PSD-95 by controlling c-Jun N-terminal kinase activity and PSD-95 phosphorylation. Here, we show that the IgG-like extracellular domains of IL1RAPL1 induce excitatory pre-synapse formation by interacting with protein tyrosine phosphatase delta (PTPδ). We also found that IL1RAPL1 TIR domains interact with RhoGAP2, which is localized at the excitatory post-synaptic density. More interestingly, the IL1RAPL1/PTPδ complex recruits RhoGAP2 at excitatory synapses to induce dendritic spine formation. We also found that the IL1RAPL1 paralog, IL1RAPL2, interacts with PTPδ and induces excitatory synapse and dendritic spine formation. The interaction of the IL1RAPL1 family of proteins with PTPδ and RhoGAP2 reveals a pathophysiological mechanism of cognitive impairment associated with a novel type of trans-synaptic signaling that regulates excitatory synapse and dendritic spine formation.

Structural insights into the assembly and activation of IL-1ß with its receptors.

Interleukin 1ß (IL-1ß) is a key orchestrator of inflammation and host defense that exerts its effects through IL-1 receptor type I (IL-1RI) and IL-1 receptor accessory protein (IL-1RAcP). How IL-1RAcP is recruited by IL-1ß-IL-1RI to form the signaling-competent complex remains elusive. Here we present the crystal structure of IL-1ß bound to IL-1 receptor type II (IL-1RII) and IL-1RAcP. IL-1ß-IL-1RII generated a composite binding surface to recruit IL-1RAcP. Biochemical analysis demonstrated that IL-1ß-IL-1RI and IL-1ß-IL-1RII interacted similarly with IL-1RAcP. It also showed the importance of two loops of IL-1 receptor antagonist (IL-1Ra) in determining its antagonism. Our results provide a structural basis for assembly and activation of the IL-1 receptor and offer a general cytokine-receptor architecture that governs the IL-1 family of cytokines.

Structure of IL-33 and its interaction with the ST2 and IL-1RAcP receptors--insight into heterotrimeric IL-1 signaling complexes.

Members of the interleukin-1 (IL-1) family of cytokines play major roles in host defense and immune system regulation in infectious and inflammatory diseases. IL-1 cytokines trigger a biological response in effector cells by assembling a heterotrimeric signaling complex with two IL-1 receptor chains, a high-affinity primary receptor and a low-affinity coreceptor. To gain insights into the signaling mechanism of the novel IL-1-like cytokine IL-33, we first solved its solution structure and then performed a detailed biochemical and structural characterization of the interaction between IL-33, its primary receptor ST2, and the coreceptor IL-1RAcP. Using nuclear magnetic resonance data, we obtained a model of the IL-33/ST2 complex in solution that is validated by small-angle X-ray scattering (SAXS) data and is similar to the IL-1beta/IL-1R1 complex. We extended our SAXS analysis to the IL-33/ST2/IL-1RAcP and IL-1beta/IL-1R1/IL-1RAcP complexes and propose a general model of the molecular architecture of IL-1 ternary signaling complexes.

A central nervous system-restricted isoform of the interleukin-1 receptor accessory protein modulates neuronal responses to interleukin-1.

Interleukin-1 (IL-1) has multiple functions in both the periphery and the central nervous system (CNS) and is regulated at many levels. We identified an isoform of the IL-1 receptor (IL-1R) accessory protein (termed AcPb) that is expressed exclusively in the CNS. AcPb interacted with IL-1 and the IL-1R but was unable to mediate canonical IL-1 responses. AcPb expression, however, modulated neuronal gene expression in response to IL-1 treatment in vitro. Animals lacking AcPb demonstrated an intact peripheral IL-1 response and developed experimental autoimmune encephalomyelitis (EAE) similarly to wild-type mice. AcPb-deficient mice were instead more vulnerable to local inflammatory challenge in the CNS and suffered enhanced neuronal degeneration as compared to AcP-deficient or wild-type mice. These findings implicate AcPb as an additional component of the highly regulated IL-1 system and suggest that it may play a role in modulating CNS responses to IL-1 and the interplay between inflammation and neuronal survival.

Autologous conditioned serum in the treatment of orthopedic diseases: the orthokine therapy.

The common strategies for the treatment of patients with orthopedic diseases do not address the underlying pathogenesis. Several biologically based, local therapies aiming to influence the cytokine imbalance are either in development or in the initial stages of clinical use. A method based on exposure of blood leukocytes to pyrogen-free surfaces (e.g. glass spheres) elicits an accumulation of anti-inflammatory cytokines, including interleukin-1 receptor antagonist, and several growth factors, including insulin-like growth factor-1, platelet-derived growth factor, and transforming growth factor-beta(1), in the liquid blood phase. Based on these observations, a new therapy using cell-free, autologous conditioned serum (ACS) from the incubation of whole blood with glass spheres was developed. The injection of ACS into affected tissue(s) has shown clinical effectiveness and safety in animal models and studies, as well as in human clinical studies, for the treatment of osteoarthritis, lumbar stenosis, disc prolapse, and muscle injuries.

Mass spectrometric analysis of the endogenous type I interleukin-1 (IL-1) receptor signaling complex formed after IL-1 binding identifies IL-1RAcP, MyD88, and IRAK-4 as the stable components.

We investigated the composition of the endogenous ligand-bound type I interleukin-1 (IL-1) receptor (IL-1RI) signaling complex using immunoprecipitation and tandem mass spectrometry. Three proteins with approximate molecular masses of 60 (p60), 36 (p36), and 90 kDa (p90) became phosphorylated after treatment with IL-1. Phosphorylation in vitro of p60 has been reported previously, but its identity was unknown. We showed using tandem mass spectrometry that p60 is identical to interleukin-1 receptor-associated kinase (IRAK)-4. MS also enabled detection of IL-1, IL-1RI, IL-1 receptor accessory protein (IL-1RAcP), and myeloid differentiation primary response protein 88 (MyD88) in the complex. The p60 protein (IRAK-4) was the earliest component of the complex to be phosphorylated. Phosphorylated IRAK-4 from the receptor complex migrated more slowly in SDS-PAGE than its unphosphorylated form as did recombinant IRAK-4 autophosphorylated in vitro. Phosphorylation was restricted to serine and threonine residues. IRAK-4, p36, IL-1RAcP, and MyD88 bound to the liganded receptor within 15 s of activation by IL-1 and remained associated upon prolonged activation, suggesting that the signaling complex is very stable. The p90 phosphoprotein was only transiently associated with the receptor. This behavior and its size were consistent with it being IRAK-1. Our work revealed that liganding of IL-1RI causes its strong and stable association with IL-1RAcP, MyD88, and the previously unidentified protein p60 (IRAK-4). The only component of the IL-1RI signaling complex that dissociated is IRAK-1. Our study is therefore the first detailed description of the endogenous IL-1RI complex.