SELEX-discovered aptamer that inhibits cellular interleukin-17/interleukin-17 receptor interaction and antagonizes interleukin-17 signaling.

This research is based on a Systematic Evolution of Ligands by EXponential enrichment, also referred to as in vitro selection against the extracellular domain of human interleukin-17 receptor A (IL-17RA). Pull-down assay via quantitative polymerase chain reaction and chemiluminescence detection showed that the cloned RNA with the enriched sequence bound to human IL-17RA and inhibited the interaction between IL-17RA and human interleukin-17A (IL-17A). We also revealed that the newly discovered IL-17RA-binding RNA aptamer bound to cellular IL-17RA, inhibited the cellular IL-17RA/IL-17A interaction, and antagonized cellular IL-17A signaling.

Molecular modeling and rational design of disulfide-stapled self-inhibitory peptides to target IL-17A/IL-17RA interaction.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine implicated in diverse autoimmune and inflammatory disorders such as psoriasis and Kawasaki disease. Mature IL-17A is a homodimer that binds to the extracellular type-III fibronectin D1:D2-dual domain of its cognate IL-17 receptor A (IL-17RA). In this study, we systematically examined the structural basis, thermodynamics property, and dynamics behavior of IL-17RA/IL-17A interaction and computationally identified two continuous hotspot regions separately from different monomers of IL-17A homodimer that contribute significantly to the interaction, namely I-shaped and U-shaped segments, thus rendered as a peptide-mediated protein-protein interaction (PmPPI). Self-inhibitory peptides (SIPs) are derived from the two segments to disrupt IL-17RA/IL-17A interaction by competitively rebinding to the IL-17A-binding pocket on IL-17RA surface, which, however, only have a weak affinity and low specificity for IL-17RA due to lack of the context support of intact IL-17A protein, thus exhibiting a large flexibility and intrinsic disorder when splitting from the protein context and incurring a considerable entropy penalty when rebinding to IL-17RA. The U-shaped segment is further extended, mutated and stapled by a disulfide bridge across its two strands to obtain a number of double-stranded cyclic SIPs, which are partially ordered and conformationally similar to their native status at IL-17RA/IL-17A complex interface. Experimental fluorescence polarization assays substantiate that the stapling can moderately or considerably improve the binding affinity of U-shaped segment-derived peptides by 2-5-fold. In addition, computational structural modeling also reveals that the stapled peptides can bind in a similar mode with the native crystal conformation of U-shaped segment in IL-17RA pocket, where the disulfide bridge is out of the pocket for avoiding intervene of the peptide binding.

Organizing structural principles of the IL-17 ligand-receptor axis.

The IL-17 family of cytokines and receptors have central roles in host defence against infection and development of inflammatory diseases1. The compositions and structures of functional IL-17 family ligand-receptor signalling assemblies remain unclear. IL-17E (also known as IL-25) is a key regulator of type 2 immune responses and driver of inflammatory diseases, such as allergic asthma, and requires both IL-17 receptor A (IL-17RA) and IL-17RB to elicit functional responses2. Here we studied IL-25-IL-17RB binary and IL-25-IL-17RB-IL-17RA ternary complexes using a combination of cryo-electron microscopy, single-molecule imaging and cell-based signalling approaches. The IL-25-IL-17RB-IL-17RA ternary signalling assembly is a C2-symmetric complex in which the IL-25-IL-17RB homodimer is flanked by two 'wing-like' IL-17RA co-receptors through a 'tip-to-tip' geometry that is the key receptor-receptor interaction required for initiation of signal transduction. IL-25 interacts solely with IL-17RB to allosterically promote the formation of the IL-17RB-IL-17RA tip-to-tip interface. The resulting large separation between the receptors at the membrane-proximal level may reflect proximity constraints imposed by the intracellular domains for signalling. Cryo-electron microscopy structures of IL-17A-IL-17RA and IL-17A-IL-17RA-IL-17RC complexes reveal that this tip-to-tip architecture is a key organizing principle of the IL-17 receptor family. Furthermore, these studies reveal dual actions for IL-17RA sharing among IL-17 cytokine complexes, by either directly engaging IL-17 cytokines or alternatively functioning as a co-receptor.

Interleukin-25 recognition by its unique receptor IL-17Rb via two discrete linear and cyclic epitopes.

Interleukin-17 (IL-17) is a family of pro-inflammatory cytokines and has been involved in the pathogenesis of chronic inflammatory and autoimmune diseases. The IL-17E, also known as IL-25, is a distinct member of this family that binds to its unique receptor IL-17Rb to induce the activation of nuclear factor kappa-light-chain enhancer of activated B cells. Here, we systematically examined the intermolecular recognition and association of IL-25 with IL-17Rb and demonstrated that the IL-25 primarily adopts two discrete linear and cyclic epitopes to interact with IL-17Rb. The two epitopes are separately located in the monomers 1 and 2 of IL-25 homodimer and cover sequences 125 DPRGNSELLYHN136 and 77 ELDRDLNRLPQDLY90 . They totally contribute 71.6% binding energy to the full-length IL-25. The linear epitope targets a site spanning over the extracellular fnIIID1 and fnIIID2 domains of IL-17Rb, while the cyclic epitope primarily binds at the fnIIID1 domain. In addition, we also found that the linear and cyclic epitopes are natively folded into ordered single-stranded and double-stranded conformations in IL-25 protein context, respectively, but would become largely disordered when splitting from the context to be free peptides, which, however, cannot bind effectively to IL-17Rb as them in the native state. In this respect, we extended the cyclic epitope to cover the whole IL-25 double-stranded region and added a disulfide bridge across its two strands at three selected anchor residue pairs. It is revealed that the disulfide-stapled peptides can be constrained into a native-like conformation and thus exhibit an improved binding potency to IL-17Rb as compared to their unstapled counterpart.

Self-assembled chitosan nanoparticles for intranasal delivery of recombinant protein interleukin-17 receptor C (IL-17RC): preparation and evaluation in asthma mice.

Asthma is mentioned as a chronic airway inflammatory disease, whose pathogenesis is complicated. The promotion of inflammation in asthma by IL-17A and IL-17F has been confirmed. In addition to covalent homodimers, both cytokines are also able to form heterodimers, further inducing downstream pathways via binding to the IL-17RA and IL-17RC receptor complex. In recent years, IL-17RA and its signal transduction pathway have been extensively researched. IL-17RC, however, remains relatively unexplored. In the present study, we self-assembled chitosan (CS) nanoparticles for intranasal delivery of recombinant protein IL-17RC (rIL-17RC) and preliminarily investigated its effect on a murine model of allergic asthma induced by ovalbumin (OVA). rIL-17RC was produced by the prokaryotic expression system and encapsulated into the CS nanoparticles via ionic cross-linking technique. The results showed that CS-RC nanoparticles via intranasal intervention significantly caused inhibition of mucus secretion and airway inflammatory cell infiltration, and reduced IL-4, IL-17, IL-17F levels in BALF. Hence, delivering receptor proteins such as IL-17RC, through CS nanoparticles as a carrier, could be an attractive therapeutic intervention for asthma.

Interleukin-17 receptor D (Sef) is a multi-functional regulator of cell signaling.

Interleukin-17 receptor D (IL17RD or IL-17RD) also known as Sef (similar expression to fibroblast growth factor), is a single pass transmembrane protein that is reported to regulate several signaling pathways . IL17RD was initially described as a feedback inhibitor of fibroblast growth factor (FGF) signaling during zebrafish and frog development. It was subsequently determined to regulate other receptor tyrosine kinase signaling cascades as well as several proinflammatory signaling pathways including Interleukin-17A (IL17A), Toll-like receptors (TLR) and Interleukin-1α (IL1α) in several vertebrate species including humans. This review will provide an overview of IL17RD regulation of signaling pathways and functions with emphasis on regulation of development and pathobiological conditions. We will also discuss gaps in our knowledge about IL17RD function to provide insight into opportunities for future investigation. Video Abstract.

Structural Analysis Reveals that the Cytokine IL-17F Forms a Homodimeric Complex with Receptor IL-17RC to Drive IL-17RA-Independent Signaling.

Interleukin-17A (IL-17A), IL-17F, and IL-17A/F heterodimers are key cytokines of the innate and adaptive immune response. Dysregulation of the IL-17 pathway contributes to immune pathology, and it is therefore important to elucidate the molecular mechanisms that govern IL-17 recognition and signaling. The receptor IL-17RC is thought to act in concert with IL-17RA to transduce IL-17A-, IL-17F-, and IL-17A/F-mediated signals. We report the crystal structure of the extracellular domain of human IL-17RC in complex with IL-17F. In contrast to the expected model, we found that IL-17RC formed a symmetrical 2:1 complex with IL-17F, thus competing with IL-17RA for cytokine binding. Using biophysical techniques, we showed that IL-17A and IL-17A/F also form 2:1 complexes with IL-17RC, suggesting the possibility of IL-17RA-independent IL-17 signaling pathways. The crystal structure of the IL-17RC:IL-17F complex provides a structural basis for IL-17F signaling through IL-17RC, with potential therapeutic applications for respiratory allergy and inflammatory bowel diseases.

Structural Insights into the Interleukin-17 Family Cytokines and Their Receptors.

The IL-17 family in humans consists of six distinct cytokines (IL-17A-F) that can interact with five IL-17 receptors (IL-17RA-E). The interaction between these cytokines and their receptors are critical in mediating host defenses while also making major contributions to inflammatory and autoimmune responses as demonstrated through both in vitro and in vivo experiments as well as human clinical trials. Inhibition of the IL-17A/IL-17RA interaction by monoclonal antibodies has also displayed remarkable efficacies in clinical trials against psoriasis and other autoimmune diseases. Recently, we and others reported the identification and characterization of both small-molecule and peptide IL-17A antagonists. These non-antibody IL-17A antagonists can effectively and selectively disrupt the IL-17A/IL-17RA complex and may provide alternative modalities to treat IL-17-related autoimmune and inflammatory diseases. This chapter summarizes the reported crystal structures of the IL-17 cytokines, their complexes with IL-17RA, and their complexes with both monoclonal antibodies as well as small-molecule and peptide antagonists.

ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis.

Interleukin 17 (IL-17) and its cognate receptor A (IL-17RA) play a crucial role in Th17 cells-mediated pro-inflammatory pathway and pathogenesis of several autoimmune disorders including psoriasis. IL-17 is mainly produced by activated Th-17 helper cells upon stimulation by IL-23 and, via binding to its receptors, mediates IL-17-driven cell signaling in keratinocytes. Hyper-proliferation of keratinocytes belongs to major clinical manifestations in psoriasis. To modulate IL-17-mediated inflammatory cascade, we generated a unique collection of IL-17RA-targeting protein binders that prevent from binding of human IL-17A cytokine to its cell-surface receptor. To this goal, we used a highly complex combinatorial library derived from scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived high-affinity ligands of human IL-17RA, called ARS binders. From 67 analyzed ABD variants, 7 different sequence families were identified. Representatives of these groups competed with human IL-17A for binding to recombinant IL-17RA receptor as well as to IL-17RA-Immunoglobulin G chimera, as tested in enzyme-linked immunosorbent assay (ELISA). Five ARS variants bound to IL-17RA-expressing THP-1 cells and blocked binding of human IL-17 cytokine to the cell surface, as tested by flow cytometry. Three variants exhibited high-affinity binding with a nanomolar Kd value to human keratinocyte HaCaT cells, as measured using Ligand Tracer Green Line. Upon IL-17-stimulated activation, ARS variants inhibited secretion of Gro-α (CXCL1) by normal human skin fibroblasts in vitro. Thus, we identified a novel class of inhibitory ligands that might serve as immunosuppressive IL-17RA-targeted non-IgG protein antagonists.

Utilization of peptide phage display to investigate hotspots on IL-17A and what it means for drug discovery.

To date, IL-17A antibodies remain the only therapeutic approach to correct the abnormal activation of the IL-17A/IL-17R signaling complex. Why is it that despite the remarkable success of IL-17 antibodies, there is no small molecule antagonist of IL-17A in the clinic? Here we offer a unique approach to address this question. In order to understand the interaction of IL-17A with its receptor, we combined peptide discovery using phage display with HDX, crystallography, and functional assays to map and characterize hot regions that contribute to most of the energetics of the IL-17A/IL-17R interaction. These functional maps are proposed to serve as a guide to aid in the development of small molecules that bind to IL-17A and block its interaction with IL-17RA.

The flavonoid cyanidin blocks binding of the cytokine interleukin-17A to the IL-17RA subunit to alleviate inflammation in vivo.

Cyanidin, a key flavonoid that is present in red berries and other fruits, attenuates the development of several diseases, including asthma, diabetes, atherosclerosis, and cancer, through its anti-inflammatory effects. We investigated the molecular basis of cyanidin action. Through a structure-based search for small molecules that inhibit signaling by the proinflammatory cytokine interleukin-17A (IL-17A), we found that cyanidin specifically recognizes an IL-17A binding site in the IL-17A receptor subunit (IL-17RA) and inhibits the IL-17A/IL-17RA interaction. Experiments with mice demonstrated that cyanidin inhibited IL-17A-induced skin hyperplasia, attenuated inflammation induced by IL-17-producing T helper 17 (TH17) cells (but not that induced by TH1 or TH2 cells), and alleviated airway hyperreactivity in models of steroid-resistant and severe asthma. Our findings uncover a previously uncharacterized molecular mechanism of action of cyanidin, which may inform its further development into an effective small-molecule drug for the treatment of IL-17A-dependent inflammatory diseases and cancer.

Structural Basis of Neurohormone Perception by the Receptor Tyrosine Kinase Torso.

In insects, brain-derived Prothoracicotropic hormone (PTTH) activates the receptor tyrosine kinase (RTK) Torso to initiate metamorphosis through the release of ecdysone. We have determined the crystal structure of silkworm PTTH in complex with the ligand-binding region of Torso. Here we show that ligand-induced Torso dimerization results from the sequential and negatively cooperative formation of asymmetric heterotetramers. Mathematical modeling of receptor activation based upon our biophysical studies shows that ligand pulses are "buffered" at low receptor levels, leading to a sustained signal. By contrast, high levels of Torso develop the signal intensity and duration of a noncooperative system. We propose that this may allow Torso to coordinate widely different functions from a single ligand by tuning receptor levels. Phylogenic analysis indicates that Torso is found outside arthropods, including human parasitic roundworms. Together, our findings provide mechanistic insight into how this receptor system, with roles in embryonic and adult development, is regulated.

Therapeutic Potential of IL-17-Mediated Signaling Pathway in Autoimmune Liver Diseases.

Emerging evidence reveals that various cytokines and tissue microenvironments contribute to liver inflammation and autoimmunity, and IL-17 family is one of highlights acknowledged. Although the implication of IL-17 family in most common autoimmune diseases (such as psoriasis, inflammatory bowel disease, and rheumatoid arthritis) has been extensively characterized, the role of this critical family in pathophysiology of autoimmune liver diseases (AILD) still needs to be clarified. In the review, we look into the intriguing biology of IL-17 family and further dissect on the intricate role of IL-17-mediated pathway in AILD. Considering encouraging data from preclinical and clinical trials, IL-17 targeted therapy has shown promises in several certain autoimmune conditions. However, blocking IL-17-mediated pathway is just beginning, and more fully investigation and reflection are required. Taking together, targeting IL-17-mediated responses may open up new areas of potential clinical treatment for AILD.

An Overview of Interleukin-17A and Interleukin-17 Receptor A Structure, Interaction and Signaling.

Interleukin-17A (IL-17A) and its receptor (IL-17RA) are prototype members of IL-17 ligand/receptor family firstly identified in CD4+ T cells, which comprises six ligands (IL-17A to IL- 17F) and five receptors (IL-17RA to IL-17RE). IL-17A is predominantly secreted by T helper 17 (Th17) cells, and plays important roles in the development of autoimmune and inflammatory diseases. IL-17RA is widely expressed, and forms a complex with IL-17RC. Binding of IL-17A to this receptor complex triggers the activation of several intracellular signaling pathways. In this review, we aimed to summarize literature data about molecular features of IL-17A and IL-17RA from gene to mature protein. We are also providing insight into regulatory mechanisms, protein structural conformation, including ligand-receptor interaction, and an overview of signaling pathways. Our aim was to compile the data on molecular characteristics of IL-17A and IL-17RA which may help in the understanding of their functions in health and disease.

IL-17RA in intestinal inflammation: structure, signaling, function, and clinical application.

: Interleukin-17 receptor A (IL-17RA) is responsible for both IL-17A and IL-25 (IL-17E) signaling pathways. Current evidences suggest distinct but interactive responses between IL-17A and IL-25 signaling, both of which are critical for intestinal immune homeostasis. IL-17RA is assumed to regulate this counterbalance and therefore becomes a crucial molecule in mucosal immunology. In this review, we will describe the structure of IL-17RA, compare IL-17A and IL-25 signaling pathways, and emphasize on the function of IL-17RA in intestinal inflammation and discuss current evidences of accomplished and ongoing clinical trials with monoclonal antibodies targeting Th17 pathway, especially IL-17RA.

Tumor necrosis factor receptor 2 (TNFR2)·interleukin-17 receptor D (IL-17RD) heteromerization reveals a novel mechanism for NF-κB activation.

TNF receptor 2 (TNFR2) exerts diverse roles in the pathogenesis of inflammatory and autoimmune diseases. Here, we report that TNFR2 but not TNFR1 forms a heteromer with interleukin-17 receptor D (IL-17RD), also named Sef, to activate NF-κB signaling. TNFR2 associates with IL-17RD, leading to mutual receptor aggregation and TRAF2 recruitment, which further activate the downstream cascade of NF-κB signaling. Depletion of IL-17RD impaired TNFR2-mediated activation of NF-κB signaling. Importantly, IL-17RD was markedly increased in renal tubular epithelial cells in nephritis rats, and a strong interaction of TNFR2 and IL-17RD was observed in the renal epithelia. The IL-17RD·TNFR2 complex in activation of NF-κB may explain the role of TNFR2 in inflammatory diseases including nephritis.

Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved α-helix for Act1 binding and IL-17 signaling.

Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein-protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand ßC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC'ins) and a flexible loop (CC'). The DD' loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Što accommodate the αCC'ins helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR-SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.

IL-17 family: cytokines, receptors and signaling.

The interleukin 17 (IL-17) family, a subset of cytokines consisting of IL-17A-F, plays crucial roles in host defense against microbial organisms and in the development of inflammatory diseases. Although IL-17A is the signature cytokine produced by T helper 17 (Th17) cells, IL-17A and other IL-17 family cytokines have multiple sources ranging from immune cells to non-immune cells. The IL-17 family signals via their correspondent receptors and activates downstream pathways that include NFκB, MAPKs and C/EBPs to induce the expression of anti-microbial peptides, cytokines and chemokines. The proximal adaptor Act1 is a common mediator during the signaling of all IL-17 cytokines so far and is thus involved in IL-17 mediated host defense and IL-17-driven autoimmune conditions. This review will give an overview and recent updates on the IL-17 family, the activation and regulation of IL-17 signaling as well as diseases associated with this cytokine family.

The anaphase-promoting complex protein 5 (AnapC5) associates with A20 and inhibits IL-17-mediated signal transduction.

IL-17 is the founding member of a family of cytokines and receptors with unique structures and signaling properties. IL-17 is the signature cytokine of Th17 cells, a relatively new T cell population that promotes inflammation in settings of infection and autoimmunity. Despite advances in understanding Th17 cells, mechanisms of IL-17-mediated signal transduction are less well defined. IL-17 signaling requires contributions from two receptor subunits, IL-17RA and IL-17RC. Mutants of IL-17RC lacking the cytoplasmic domain are nonfunctional, indicating that IL-17RC provides essential but poorly understood signaling contributions to IL-17-mediated signaling. To better understand the role of IL-17RC in signaling, we performed a yeast 2-hybrid screen to identify novel proteins associated with the IL-17RC cytoplasmic tail. One of the most frequent candidates was the anaphase promoting complex protein 7 (APC7 or AnapC7), which interacted with both IL-17RC and IL-17RA. Knockdown of AnapC7 by siRNA silencing exerted no detectable impact on IL-17 signaling. However, AnapC5, which associates with AnapC7, was also able to bind IL-17RA and IL-17RC. Moreover, AnapC5 silencing enhanced IL-17-induced gene expression, suggesting an inhibitory activity. Strikingly, AnapC5 also associated with A20 (TNFAIP3), a recently-identified negative feedback regulator of IL-17 signal transduction. IL-17 signaling was not impacted by knockdown of Itch or TAXBP1, scaffolding proteins that mediate A20 inhibition in the TNFα and IL-1 signaling pathways. These data suggest a model in which AnapC5, rather than TAX1BP1 and Itch, is a novel adaptor and negative regulator of IL-17 signaling pathways.

Crystal structures of interleukin 17A and its complex with IL-17 receptor A.

The constituent polypeptides of the interleukin-17 family form six different homodimeric cytokines (IL-17A-F) and the heterodimeric IL-17A/F. Their interactions with IL-17 receptors A-E (IL-17RA-E) mediate host defenses while also contributing to inflammatory and autoimmune responses. IL-17A and IL-17F both preferentially engage a receptor complex containing one molecule of IL-17RA and one molecule of IL-17RC. More generally, IL-17RA appears to be a shared receptor that pairs with other members of its family to allow signaling of different IL-17 cytokines. Here we report crystal structures of homodimeric IL-17A and its complex with IL-17RA. Binding to IL-17RA at one side of the IL-17A molecule induces a conformational change in the second, symmetry-related receptor site of IL-17A. This change favors, and is sufficient to account for, the selection of a different receptor polypeptide to complete the cytokine-receptor complex. The structural results are supported by biophysical studies with IL-17A variants produced by site-directed mutagenesis.