A modular interface of IL-4 allows for scalable affinity without affecting specificity for the IL-4 receptor

BACKGROUND: Interleukin 4 (IL-4) is a key regulator of the immune system and an important factor in the development of allergic hypersensitivity. Together with interleukin 13 (IL-13), IL-4 plays an important role in exacerbating allergic and asthmatic symptoms. For signal transduction, both cytokines can utilise the same receptor, consisting of the IL-4Rα and the IL-13Rα1 chain, offering an explanation for their overlapping biological functions. Since both cytokine ligands share only moderate similarity on the amino acid sequence level, molecular recognition of the ligands by both receptor subunits is of great interest. IL-4 and IL-13 are interesting targets for allergy and asthma therapies. Knowledge of the binding mechanism will be important for the generation of either IL-4 or IL-13 specific drugs. RESULTS: We present a structure/function analysis of the IL-4 ligand-receptor interaction. Structural determination of a number of IL-4 variants together with in vitro binding studies show that IL-4 and its high-affinity receptor subunit IL-4Rα interact via a modular protein-protein interface consisting of three independently-acting interaction clusters. For high-affinity binding of wild-type IL-4 to its receptor IL-4Rα, only two of these clusters (i.e. cluster 1 centered around Glu9 and cluster 2 around Arg88) contribute significantly to the free binding energy. Mutating residues Thr13 or Phe82 located in cluster 3 to aspartate results in super-agonistic IL-4 variants. All three clusters are fully engaged in these variants, generating a three-fold higher binding affinity for IL-4Rα. Mutagenesis studies reveal that IL-13 utilizes the same main binding determinants, i.e. Glu11 (cluster 1) and Arg64 (cluster 2), suggesting that IL-13 also uses this modular protein interface architecture. CONCLUSION: The modular architecture of the IL-4-IL-4Rα interface suggests a possible mechanism by which proteins might be able to generate binding affinity and specificity independently. So far, affinity and specificity are often considered to co-vary, i.e. high specificity requires high affinity and vice versa. Although the binding affinities of IL-4 and IL-13 to IL-4Rα differ by a factor of more than 1000, the specificity remains high because the receptor subunit IL-4Rα binds exclusively to IL-4 and IL-13. An interface formed by several interaction clusters/binding hot-spots allows for a broad range of affinities by selecting how many of these interaction clusters will contribute to the overall binding free energy. Understanding how proteins generate affinity and specificity is essential as more and more growth factor receptor families show promiscuous binding to their respective ligands. This limited specificity is, however, not accompanied by low binding affinities

Apicidin F: characterization and genetic manipulation of a new secondary metabolite gene cluster in the rice pathogen Fusarium fujikuroi.

The fungus F. fujikuroi is well known for its production of gibberellins causing the 'bakanae' disease of rice. Besides these plant hormones, it is able to produce other secondary metabolites (SMs), such as pigments and mycotoxins. Genome sequencing revealed altogether 45 potential SM gene clusters, most of which are cryptic and silent. In this study we characterize a new non-ribosomal peptide synthetase (NRPS) gene cluster that is responsible for the production of the cyclic tetrapeptide apicidin F (APF). This new SM has structural similarities to the known histone deacetylase inhibitor apicidin. To gain insight into the biosynthetic pathway, most of the 11 cluster genes were deleted, and the mutants were analyzed by HPLC-DAD and HPLC-HRMS for their ability to produce APF or new derivatives. Structure elucidation was carried out be HPLC-HRMS and NMR analysis. We identified two new derivatives of APF named apicidin J and K. Furthermore, we studied the regulation of APF biosynthesis and showed that the cluster genes are expressed under conditions of high nitrogen and acidic pH in a manner dependent on the nitrogen regulator AreB, and the pH regulator PacC. In addition, over-expression of the atypical pathway-specific transcription factor (TF)-encoding gene APF2 led to elevated expression of the cluster genes under inducing and even repressing conditions and to significantly increased product yields. Bioinformatic analyses allowed the identification of a putative Apf2 DNA-binding ("Api-box") motif in the promoters of the APF genes. Point mutations in this sequence motif caused a drastic decrease of APF production indicating that this motif is essential for activating the cluster genes. Finally, we provide a model of the APF biosynthetic pathway based on chemical identification of derivatives in the cultures of deletion mutants

Chronic viral infections in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

Background and main text: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex and controversial clinical condition without having established causative factors. Increasing numbers of cases during past decade have created awareness among patients as well as healthcare professionals. Chronic viral infection as a cause of ME/CFS has long been debated. However, lack of large studies involving well-designed patient groups and validated experimental set ups have hindered our knowledge about this disease. Moreover, recent developments regarding molecular mechanism of pathogenesis of various infectious agents cast doubts over validity of several of the past studies. Conclusions: This review aims to compile all the studies done so far to investigate various viral agents that could be associated with ME/CFS. Furthermore, we suggest strategies to better design future studies on the role of viral infections in ME/CFS

Regulation of the apicidin F cluster.

<p>(A) The pH regulator PacC seems to be an activator of the apicidin F genes. The WT and Δ<i>PACC</i> were grown for three days under optimal conditions (60 mM glutamine, gln). The cultures were harvested and after washing, the mycelium was shifted into new flasks containing 60 mM gln adjusted to an ambient pH of 4 or 8, respectively. After 2 h the cultures were harvested again. (B) The nitrogen regulators AreB and glutamine synthetase (GS) are activators of the apicidin F gene expression. The WT, Δ<i>AREA</i> and Δ<i>AREB</i> and the gln auxotroph mutant Δ<i>GLN1</i> were grown for three days in 60 mM gln. (C) The WT, Δ<i>VEL1</i>, Δ<i>VEL2</i> and Δ<i>LAE1</i> were grown for three days in 60 mM gln. <i>APF6</i> and <i>APF9</i> were used as probes for all Northern blot analyses.</p

Mutation of the putative “Api-box” motif in the promoter region of <i>APF1</i> (NRPS) and <i>APF11</i> (transporter) resulted in reduced production of apicidin F.

<p>(A) Bioinformatic searches revealed an eight-base-pair motif with the consensus sequence 5′-TGACGTGA-3′ that was found in all promoters of the apicidin F cluster except in the promoter region of the transcription factor (TF)-encoding gene itself. In our study, we created two mutants with point mutations in the <i>APF1</i>/<i>APF11</i> promoter (P-mut1 and P-mut2, for the strategy see Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336.s001" target="_blank">File S1</a>). (B) Biosynthesis of apicidin F was monitored with HPLC-HRMS. After growth for three days in 60 mM glutamine, the cultures of the WT and the two mutants P-mut1 and P-mut2 were harvested. Apicidin F was extracted from lyophilized mycelium. 10 µL of a 1 µg/mL apicidin solution (internal standard) were added to 90 µL of the sample. For the calculation, the peak area of apicidin F [M+H]⁺ (646.3235±0.0032) was divided with that of apicidin [M+H]⁺ (624.3756±0.0032). Product formation was normalized to the WT level. Experiment was performed in a triplicate.</p

Deletion of the cluster genes <i>APF3</i> and <i>APF9</i> revealed new analogs of the apicidin F biosynthetic pathway.

<p>(A) HPLC-HRMS-chromatograms of the culture filtrates of the WT and the single deletion mutants of <i>APF3</i> (Δ<i>APF3</i>) and <i>APF9</i> (Δ<i>APF9</i>) grown in ICI with 60 mM glutamine for three days. Shown are the extracted ion chromatograms for the [M+H]⁺-ion of proline apicidin F (apicidin J, 632.3079±0.0032, left) and for the [M+H]⁺-ion the Δ<i>APF9</i>-product (apicidin K, 632.3443±0.0032, right). The axes are normalized to the WT-level. (B) Structures of the two identified products: apicidin J and apicidin K.</p

The transcription factor (TF) Apf2 contains a basic DNA binding domain, four ankyrin repeats and is localized in the nucleus.

<p>(A) ClustalW alignment with amino acids of <i>Cochliobolus carbonum</i> ToxE (AFO38874), <i>Fusarium semitectum</i> Aps2 (GQ331953) and <i>Fusarium fujikuroi</i> Apf2 (FFUJ_00012). Identical amino acids are highlighted in grey, the positions of the domains are highlighted in either orange (basic DNA binding domain) or green (four ankyrin repeats) and based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Pedley1" target="_blank">[55]</a>. (B) The TF was fused to green fluorescent protein (GFP) at the C-terminus. The Δ<i>APF2</i> mutant was used as background. The two strains were grown for one day in 60 mM glutamine. Size of scale bars is indicated. A supplemental figure with controls is depicted in Fig. S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336.s001" target="_blank">File S1</a>.</p

Gene name, accession numbers, length and predicted function of the apicidin F cluster and its border gene.

<p>Gene name, accession numbers, length and predicted function of the apicidin F cluster and its border gene.</p