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.

Molecular cloning, expression analysis of interleukin 17D (cysteine knot cytokine) from Amphiprion clarkii and their functional characterization and NFκB pathway activation using FHM cells.

Interleukin 17D (IL-17D), a pro-inflammatory cytokine, is a signature cytokine of T helper 17 (Th17) cells. However, studies characterizing the functions of IL-17D in teleost are scarce. Therefore, we aimed to characterize the properties of IL-17D in Amphiprion clarkii. We performed spatial and temporal expression, AcIL-17D-mediated antibacterial and inflammatory gene expression, NFκB pathway-related gene expression analyses, and bacterial colony counting and cell protection assays. We found that AcIL-17D contains a 630 bp coding sequence and encodes 210 amino acids. The spatial expression analysis of AcIL-17D in 12 tissues showed ubiquitous expression, with the highest expression in the brain, followed by blood and skin. Temporal expression analysis of AcIL-17D in blood showed upregulated expression at 6 and 24 h (polyinosinic: polycytidylic acid and lipopolysaccharide), 12 h (all stimulants), and 48 h (polyinosinic: polycytidylic acid and Vibrio harveyi). AcIL-17D expression in the blood gradually decreased at later hours in response to all the stimulants. After treatment of fathead minnow (FHM) cells with different recombinant AcIL-17D concentrations, the downstream gene expression analysis showed increased expression of antimicrobial genes in the FHM cells, namely [NK-Lysin (NKL), Hepcidin antimicrobial peptide-1 (HAMP-1), Defensin-ß (DEFB1)] and some inflammatory genes such as IL-1ß, TNF-α, IL-11, and STAT3. Further nuclear factor κB (NFκB) subunits (NFκB1, NFκB2, RelA, and Rel-B) showed upregulated gene expression at 12 and 24 h. The bacterial colony counting assay using FHM cells showed lower bacterial colony counts in rAcIL-17D-treated cells than in control. Furthermore, the Water-Soluble Tetrazolium Salt (WST -1) assay confirmed the ability of rAcIL-17D in the protection of FHM cells from bacterial infection and conducted the Hoechst 33342 staining upon treatment with rAcIL-17D and rMBP. Therefore, our findings provide important insights into the activation of IL-17D pathway genes in FHM cells, the protective role of AcIL-17D against bacterial infection, and host defense mechanisms in teleost.

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.

Implementing Curcumin in Translational Oncology Research.

Most data published on curcumin and curcumin-based formulations are very promising. In cancer research, the majority of data has been obtained in vitro. Less frequently, researchers used experimental animals. The results of several clinical studies are conclusive, and these studies have established a good foundation for further research focusing on implementing curcumin in clinical oncology. However, the issues regarding timely data reporting and lack of disclosure of the exact curcumin formulations used in these studies should not be neglected. This article is a snapshot of the current status of publicly available data on curcumin clinical trials and a detailed presentation of results obtained so far with some curcumin formulations. Phenomena related to the observed effects of curcumin shown in clinical trials are presented, and its modifying effect on gut microbiota and metabolic reprogramming is discussed. Based on available data, there is a strong indication that curcumin and its metabolites present molecules that do not necessarily need to be abundant in order to act locally and benefit systemically. Future clinical studies should be designed in a way that will take that fact into consideration.

Protein-protein interactions: a structural view of inhibition strategies and the IL-23/IL-17 axis.

Protein-protein interactions are central to biology and provide opportunities to modulate disease with small-molecule or protein therapeutics. Recent developments in the understanding of the tractability of protein-protein interactions are discussed with a focus on the ligandable nature of protein-protein interaction surfaces. General principles of inhibiting protein-protein interactions are illustrated with structural biology examples from six members of the IL-23/IL-17 signaling family (IL-1, IL-6, IL-17, IL-23 RORγT and TNFα). These examples illustrate the different approaches to discover protein-protein interaction inhibitors on a target-specific basis that has proven fruitful in terms of discovering both small molecule and biologic based protein-protein interaction inhibitors.

Sequence characterization and expression pattern analysis of six kinds of IL-17 family genes in the Asian swamp eel (Monopterus albus).

Interleukin-17 (IL-17) is an important cytokine that plays a critical role in the inflammatory response and host defense against extracellular pathogens. In the present study, six novel IL-17 family genes (MaIL-17) were identified by analyzing Asian swamp eel (Monopterus albus) genome. Sequence analysis revealed that the MaIL-17 family genes shared similar features, comprising a signal peptide, an IL-17 superfamily region, and four conserved cysteines. Phylogenetic analysis showed that the MaIL-17 genes were clustered together with their corresponding IL-17 genes from other species. The similarity and identity of all IL-17 family genes indicated that the MaIL-17 genes are conserved among teleosts, while Ma-IL-17D is more conserved than the other Ma-IL-17s. Except for MaIL-17A/F3 and MaIL-17D, all MaIL-17s shared the same genomic structure as the genes from other fish, namely three exons and two introns. The MaIL-17s showed conserved synteny among fish, and we found that the MaIL-17D locus has a more conserved syntenic relationship with the loci from other fish and humans. These results demonstrated that MaIL-17D and human IL-17D might have evolved from a common ancestral gene and subsequently diverged. The analysis of swamp eel reference genes revealed that EEF1A1 (encoding eukaryotic translation elongation factor 1 alpha 1) was an ideal reference gene for accurate real-time qRT-PCR normalization in the swamp eel. The MaIL-17 genes are widely distributed throughout tissues, suggesting that MaIL-17s carry out their biological functions in immune and non-immune tissues compartments. The transcript of Ma-IL17s exhibited different fold changes in head kidney cells in response to Aeromonas veronii phorbol 12-myristate 13-acetate (PMA) and polyinosinic:polycytidylic acid (poly I:C) challenge, showing that MaIL-17A/F1 has stronger antiviral activities compared with other MaIL-17 family genes, and that MaIL-17A/F3 and MaIL-17A/F2 possess stronger effects against extracellular pathogens compared with the others; however, MaIL-17C2 and MaIL-17D may play vital roles during pathogen infection. The differential immune responses of these genes to Aeromonas veronii, PMA and poly I:C implied distinct mechanisms of host defense against extracellular pathogens.

Inhibition of CXCR4 by MicroRNA-1192 Reduces the Activation of Th17 Cells and Expression of Inflammation Factors in a Mouse Model of Vulvovaginal Candidiasis.

BACKGROUND/AIMS: Vulvovaginal candidiasis (VVC) is a disease commonly occurring in sexually active women. The involvement of microRNAs in several kinds of infectious diseases has been highlighted in a number of researches. Therefore, we conducted the present study in order to investigate whether microRNA-1192 (miR-1192) would significantly target CXCR4 in Th17 cells as well as inflammatory factors in mouse models suffering from VVC. METHODS: Seventy-five mice were selected as test subjects for this study, of which twenty-five were used as the normal control, while the rest were treated with estradiol or oil-treated in order to establish VVC mouse models (each n = 25). Protein expressions of CXCR4, IL-6, IL-17, and IL-23 were all measured using both an immunohistochemistry and ELISA. The Th17 cell percentage in peripheral blood and the expression of RORγt in Th17 cells were detected using a flow cytometry. Mouse vaginal epithelial cells were isolated from normal mice, after which the mice were treated with estradiol to regulate their estrogen, followed by treatments involving the miR-1192 mimic, miR-1192 inhibitor, siRNA-CXCR4, and miR-1192 inhibitor + si-CXCR4. The cell cycle, apoptosis, and proliferation were all examined by using an additional flow cytometry as well as the employment of the MTT assay. The miR-1192, CXCR4, IL-6, IL-17, and IL-23 expressions in tissues and cells were both measured using both RT-qPCR and western blot assay techniques. RESULTS: The mice treated with either estradiol or oil had presented to us lowered levels in miR-1192 expression as well as higher levels in both Th17 cell percentage and expression of RORγt in Th17 cells, along with mRNA and protein expressions of CXCR4, IL-6, IL-17, and IL-23. In cell experiments, the mouse vaginal epithelial cells that had been treated with miR-1192 inhibitor had shown us a decreased cell proliferation rate and contrarily increased expressions of CXCR4, IL-6, IL-17, and IL-23 mRNA, protein, and cell apoptosis rate; these results were opposite to the ones found in the mice treated with miR-1192 mimic. CONCLUSION: Our results provided significant evidence that miR-1192 could directly development and progression of VVC by restraining the CXCR4 gene in the VVC mice.

Interleukin 17 enhances bone morphogenetic protein-2-induced ectopic bone formation.

Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells in vitro through a synergy with bone morphogenetic protein (BMP)-2. This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced bone formation. Since IL-17 is known to induce osteoclastogenesis, we studied the interactions between IL-17 and BMP-2 in ceramic scaffolds either or not carrying a coating with the bisphosphonate zoledronic acid (ZOL). Histological evaluation revealed that IL-17 alone did not induce any osteoclasts at day 10. On the other hand, BMP-2 clearly stimulated early tissue ingrowth and osteoclastogenesis. Both of these processes were blocked in presence of ZOL. IL-17 signaling restored early vascularized connective tissue formation and osteoclastogenesis induced by BMP-2 in ZOL-coated scaffolds. After 12 weeks, the bone volume induced by co-delivery of BMP-2 and IL-17 was doubled as compared to that induced by BMP-2 alone. We conclude that IL-17 has osteo-stimulatory effects through a synergy with bone-inductive BMP-2. Although local and single application of IL-17 does not mediate osteoclast formation, it could promote other processes involved in bone formation such as connective tissue ingrowth. The use of IL-17 may contribute to the development of improved bone graft substitutes.

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.

The assessment of host and bacterial proteins in sputum from active pulmonary tuberculosis.

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis. The protein composition of sputum may reflect the immune status of the lung. This study aimed to evaluate the protein profiles in spontaneous sputum samples from patients with active pulmonary TB. Sputum samples were collected from patients with pulmonary TB and healthy controls. Western blotting was used to analyze the amount of interleukin 10 (IL-10), interferon-gamma (IFN-γ), IL-25, IL-17, perforin-1, urease, albumin, transferrin, lactoferrin, adenosine deaminase (also known as adenosine aminohydrolase, or ADA), ADA-2, granzyme B, granulysin, and caspase-1 in sputum. Results of detection of IL-10, IFN-γ, perforin-1, urease, ADA2, and caspase-1, showed relatively high specificity in distinguishing patients with TB from healthy controls, although sensitivities varied from 13.3% to 66.1%. By defining a positive result as the detection of any two proteins in sputum samples, combined use of transferrin and urease as markers increased sensitivity to 73.2% and specificity to 71.1%. Furthermore, we observed that the concentration of transferrin was proportional to the number of acid-fast bacilli detected in sputum specimens. Detection of sputum transferrin and urease was highly associated with pulmonary TB infection. In addition, a high concentration of transferrin detected in sputum might correlate with active TB infection. This data on sputum proteins in patients with TB may aid in the development of biomarkers to assess the severity of pulmonary TB.

Enabling N-to-C Ser/Thr Ligation for Convergent Protein Synthesis via Combining Chemical Ligation Approaches.

In this article, Ser/Thr ligation(on/off) has been realized to enable N-to-C successive peptide ligations using a salicylaldehyde semicarbazone (SAL(off)) group by in situ activation with pyruvic acid of the peptide SAL(off) ester into the peptide salicylaldehyde (SAL(on)) ester. In addition, a peptide with a C-terminal thioester and N-terminal Ser or Thr as the middle peptide segment can undergo one-pot Ser/Thr ligation and native chemical ligation in the N-to-C direction. The utility of this combined ligation strategy in the N-to-C direction has been showcased through the convergent assembly of a human cytokine protein sequence, GlcNAcylated interleukin-25.

Secukinumab for rheumatology: development and its potential place in therapy.

Rheumatic disease is not a single disorder, but a group of more than 100 diseases that affect joints, connective tissues, and/or internal organs. Although rheumatic diseases like rheumatoid arthritis (RA), psoriatic arthritis, and ankylosing spondylitis (AS) differ in their pathogenesis and clinical presentation, the treatment of these inflammatory disorders overlaps. Non-steroid anti-inflammatory drugs are used to reduce pain and inflammation. Additional disease-modifying anti-rheumatic drugs are prescribed to slowdown disease progression, and is in RA more frequently and effectively applied than in AS. Biologicals are a relatively new class of treatments that specifically target cytokines or cells of the immune system, like tumor necrosis factor alpha inhibitors or B-cell blockers. A new kid on the block is the interleukin-17 (IL-17) inhibitor secukinumab, which has been recently approved by the US Food and Drug Administration for moderate-to-severe plaque psoriasis, psoriatic arthritis, and AS. IL-17 is a proinflammatory cytokine that has an important role in host defense, but its proinflammatory and destructive effects have also been linked to pathogenic processes in autoimmune diseases like RA and psoriasis. Animal models have greatly contributed to further insights in the potential of IL-17 blockade in autoimmune and autoinflammatory diseases, and have resulted in the development of various potential drugs targeting the IL-17 pathway. Secukinumab (AIN457) is a fully human monoclonal antibody that selectively binds to IL-17A and recently entered the market under the brand name Cosentyx(®). By binding to IL-17A, secukinumab prevents it from binding to its receptor and inhibits its ability to trigger inflammatory responses that play a role in the development of various autoimmune diseases. With secukinumab being the first in class to receive Food and Drug Administration approval, this article will further focus on this new biologic agent and review the milestones in its development and marketing.

Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide.

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a ß-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.

Comparative study of four interleukin 17 cytokines of tongue sole Cynoglossus semilaevis: Genomic structure, expression pattern, and promoter activity.

The interleukin (IL)-17 cytokine family participates in the regulation of many cellular functions. In the present study, we analyzed the genomic structure, expression, and promoter activity of four IL-17 members from the teleost fish tongue sole (Cynoglossus semilaevis), i.e. CsIL-17C CsIL-17D, CsIL-17F, and IL-17F like (IL-17Fl). We found that CsIL-17C, CsIL-17D, CsIL-17F, and CsIL-17Fl share 21.2%-28.6% overall sequence identities among themselves and 31.5%-71.2% overall sequence identities with their counterparts in other teleost. All four CsIL-17 members possess an IL-17 domain and four conserved cysteine residues. Phylogenetic analysis classified the four CsIL-17 members into three clusters. Under normal physiological conditions, the four CsIL-17 expressed in multiple tissues, especially non-immune tissues. Bacterial infection upregulated the expression of all four CsIL-17, while viral infection upregulated the expression of CsIL-17D and CsIL-17Fl but downregulated the expression of CsIL-17C and CsIL-17F. The 1.2 kb 5'-flanking regions of the four CsIL-17 exhibited apparent promoter activity and contain a number of putative transcription factor-binding sites. Furthermore, the promoter activities of CsIL-17C, CsIL-17D, and CsIL-17F, but not CsIL-17Fl, were modulated to significant extents by lipopolysaccharide, PolyI:C, and PMA. This study provides the first evidence that in teleost, different IL-17 members differ in expression pattern and promoter activity.

Evolutionary Insights into IL17A in Lagomorphs.

In leporids, IL17A had been implicated in the host defense against extracellular pathogens, such as Francisella tularensis that infects hares and rabbits and causes the zoonotic disease tularemia. Here, we studied IL17A from five lagomorphs, European rabbit, pygmy rabbit, brush rabbit, European brown hare, and American pika. We observed that this protein is highly conserved between these species, with a similarity of 97-99% in leporids and ~88% between leporids and American pika. The exon/intron structure, N-glycosylation sites, and cysteine residues are conserved between lagomorphs. However, at codon 88, one of the interaction sites between IL17A and its receptor IL17RA, there is an Arg>Pro mutation that only occurs in European rabbit and European brown hare. This could induce critical alterations in the IL17A structure and conformation and consequently modify its function. The differences observed between leporids and humans or rodents might also represent important alterations in protein structure and function. In addition, as for other interleukins, IL17A sequences of human and European rabbit are more closely related than the sequences of human and mouse or European rabbit and mouse. This study gives further support to the hypothesis that European rabbit might be a more suitable animal model for studies on human IL17.

Genomic characterization and expression analysis of five novel IL-17 genes in the Pacific oyster, Crassostrea gigas.

Interleukin-17 (IL-17) is a proinflammatory cytokine that plays an important role in clearing extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. In the present study, five novel IL-17 homologs were identified by searching and analyzing the Pacific oyster genome. All six CgIL-17 members (including a previously reported homolog) contained four conserved cysteines that were used in the formation of disulfide bonds. Phylogenetic analysis showed that all invertebrate IL-17s were clustered into one group, implying that invertebrate IL-17s evolved from one common ancestral gene and subsequently diversified. All CgIL-17s shared the same genomic structure, containing two exons and one intron, except for the CgIL-17-3 and CgIL-17-5 genes, which each had only one exon. The expression pattern of the CgIL-17 genes was analyzed by qRT-PCR in a variety of tissues and at different developmental stages, and these genes were highly expressed in the gill and digestive gland tissues. Moreover, the expression of the CgIL-17 family genes was significantly up-regulated in hemocytes challenged with Pathogen-Associated Molecular Patterns (PAMPs). CgIL-17-3 had a strong response to lipopolysaccharide (LPS) and heat-killed Vibrio alginolyticus (HKVA) challenge, while CgIL-17-5 and CgIL-17-6 can be activated by peptidoglycan (PGN), but not by heat-killed Listeria monocytogenes (HKLM). The distinct, up-regulated transcript levels of the CgIL-17s in response to PAMPs challenge further indicate that CgIL-17s are likely to be significant components of immune responses by playing diversified roles in host defense in the Pacific oyster. These findings suggest that CgIL-17s are involved in innate immune responses and further supports their conserved function in mollusks immunity.

The role of interchain disulfide bond in a recombinant human interleukin-17A variant.

Interleukin-17A (IL-17A) is the prototype of IL-17 family and has been implicated in the pathogenesis of a variety of autoimmune diseases. Therefore its structural and functional properties are of great medical interest. During our research on a recombinant human IL-17A (rhIL-17A) variant, four isoforms were obtained when it was refolded. While isoforms 1 and 2 represented non-covalent dimers, isoforms 3 and 4 were determined to be covalent dimers. All four isoforms were structurally similar by Circular Dichroism and fluorescence spectroscopy studies, but differential scanning calorimetry demonstrated thermal stability in the order of isoform 1=isoform 2

Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris.

Interleukin (IL)-25 (also known as IL-17E) is a distinct member of the IL-17 cytokine family which induces IL-4, IL-5, and IL-13 expression and promotes pathogenic T helper (Th)-2 cell responses in various organs. IL-25 has been shown to have crucial role between innate and adaptive immunity and also a key component of the protection of gastrointestinal helminthes. In this study, to produce bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was performed codon optimization based on methylotropic yeast Pichia pastoris codon bias and cloned into the expression vector pPICZαA. The recombinant vector was transformed into P. pichia strain X-33 and selected by zeocin resistance. Benchtop fermentation and simple purification strategy were established to purify the rhIL-25 with about 17 kDa molecular mass. Functional analysis showed that purified rhIL-25 specifically bond to receptor IL-17BR and induce G-CSF production in vitro. Further annexin V-FITC/PI staining assay indicated that rhIL-25 induced apoptosis in two breast cancer cells, MDA-MB-231 and HBL-100. This study provides a new strategy for the large-scale production of bioactive IL-25 for biological and therapeutic applications.

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.