Joachim Grötzinger

The IL-6/sIL-6R complex as a novel target for therapeutic approaches.

IL-6 plays a pivotal role in immune responses and certain oncologic conditions. The intense investigation of its biological activity and function led to the discovery of two different IL-6-driven signalling pathways. Binding to the membrane-bound IL-6 receptor (mIL-6R, CD126) causes the recruitment of two gp130 co-receptor molecules (CD130) and the activation of intracellular signalling cascades via gp130. Although this classical pathway is mainly limited to hepatocytes, neutrophils, monocytes/macrophages and certain other leukocyte populations, which express IL-6R on their surface, an alternative mechanism has also been described. Proteolytic cleavage of the mIL-6R protein or translation from alternatively spliced mRNA leads to the generation of a soluble form of the IL-6R (sIL-6R), which is likewise able to bind to IL-6. The resulting IL-6/sIL-6R complex is also capable of binding to gp130 and inducing intracellular signalling. Through this so-called ‘trans-signalling’ mechanism, IL-6 is able to stimulate cells that lack an endogenous mIL-6R. High levels of IL-6 and sIL-6R have been reported in several chronic inflammatory and autoimmune diseases as well as in cancer. Preclinical animal disease models have provided strong evidence that specific blockade of IL-6-regulated signalling pathways represents a promising approach for the therapy of these diseases. An optimised variant of the recently described fusion protein sgp30Fc is now heading towards its clinical evaluation.

Molecular determinants of KCNQ (Kv7) K+ channel sensitivity to the anticonvulsant retigabine.

Epilepsy is caused by an electrical hyperexcitability in the CNS. Because K+ channels are critical for establishing and stabilizing the resting potential of neurons, a loss of K+ channels could support neuronal hyperexcitability. Indeed, benign familial neonatal convulsions, an autosomal dominant epilepsy of infancy, is caused by mutations in KCNQ2 or KCNQ3 K+ channel genes. Because these channels contribute to the native muscarinic-sensitive K+ current (M current) that regulates excitability of numerous types of neurons, KCNQ (Kv7) channel activators would be effective in epilepsy treatment. A compound exhibiting anticonvulsant activity in animal seizure models is retigabine. It specifically acts on the neuronally expressed KCNQ2-KCNQ5 (Kv7.2-Kv7.5) channels, whereas KCNQ1 (Kv7.1) is not affected. Using the differential sensitivity of KCNQ3 and KCNQ1 to retigabine, we constructed chimeras to identify minimal segments required for sensitivity to the drug. We identified a single tryptophan residue within the S5 segment of KCNQ3 and also KCNQ2, KCNQ4, and KCNQ5 as crucial for the effect of retigabine. Furthermore, heteromeric KCNQ channels comprising KCNQ2 and KCNQ1 transmembrane domains (attributable to transfer of assembly properties from KCNQ3 to KCNQ1) are retigabine insensitive. Transfer of the tryptophan into the KCNQ1 scaffold resulted in retigabine-sensitive heteromers, suggesting that the tryptophan is necessary in all KCNQ subunits forming a functional tetramer to confer drug sensitivity.

Soluble Interleukin-15 Receptor α (IL-15Rα)-sushi as a Selective and Potent Agonist of IL-15 Action through IL-15Rβ/γ HYPERAGONIST IL-15·IL-15Rα FUSION PROTEINS

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

Plasticity and cross-talk of Interleukin 6-type cytokines

Interleukin (IL)-6-type cytokines are critically involved in health and disease. The duration and strength of IL-6-type cytokine-mediated signaling is tightly regulated to avoid overshooting activities. Here, molecular mechanisms of inter-familiar cytokine cross-talk are reviewed which regulate dynamics and strength of IL-6 signal transduction. Both plasticity and cytokine cross-talk are significantly involved in pro- and anti-inflammatory/regenerative properties of IL-6-type cytokines. Furthermore, we focus on IL-6-type cytokine/cytokine receptor plasticity and cross-talk exemplified by the recently identified composite cytokines IL-30/IL-6R and IL-35, the first inter-familiar IL-6/IL-12 family member. The complete understanding of the intra- and extracellular cytokine networks will aid to develop novel tailor-made therapeutic strategies with reduced side effects.

Uncovering the evolutionary history of innate immunity: The simple metazoan Hydra uses epithelial cells for host defence

Although many properties of the innate immune system are shared among multicellular animals, the evolutionary origin remains poorly understood. Here we characterize the innate immune system in Hydra, one of the simplest multicellular animals known. In the complete absence of both protective mechanical barriers and mobile phagocytes, Hydras epithelium is remarkably well equipped with potent antimicrobial peptides to prevent pathogen infection. Induction of antimicrobial peptide production is mediated by the interaction of a leucine-rich repeats (LRRs) domain containing protein with a TIR-domain containing protein lacking LRRs. Conventional Toll-like receptors (TLRs) are absent in the Hydra genome. Our findings support the hypothesis that the epithelium represents the ancient system of host defence.

Soluble IL-15Rα sushi as a selective and potent agonist of IL-15 action through IL-15Rβ/γ: hyper-agonist IL-15-IL-15Rα fusion proteins

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

Benzoylformate Decarboxylase from Pseudomonas putida as Stable Catalyst for the Synthesis of Chiral 2-Hydroxy Ketones

The thiamin diphosphate- and Mg2+-dependent enzyme benzoylformate decarboxylase (BFD) from Pseudomonas putida was characterized with respect to its suitability to catalyze the formation of chiral 2-hydroxy ketones in a benzoin-condensation type reaction. Carboligation constitutes a side reaction of BFD, whereas the predominant physiological task of the enzyme is the non-oxidative decarboxylation of benzoylformate. For this purpose the enzyme was obtained in sufficient purity from Pseudomonas putida cells in a one-step purification using anion-exchange chromatography. To facilitate the access to pure BFD for kinetical studies, stability investigations, and synthetical applications, the coding gene was cloned into a vector allowing the expression of a hexahistidine fusion protein. The recombinant enzyme shows distinct activity maxima for the decarboxylation and the carboligation beside a pronounced stability in a broad pH and temperature range. The enzyme accepts a wide range of donor aldehyde substrates which are ligated to acetaldehyde as an acceptor in mostly high optical purities. The enantioselectivity of the carboligation was found to be a function of the reaction temperature, the substitution pattern of the donor aldehyde and, most significantly, of the concentration of the donor aldehyde substrate. Our data are consistent with a mechanistical model based on the X-ray crystallographic data of BFD. Furthermore we present a simple way to increase the enantiomeric excess of (S)-2-hydroxy-1-phenyl-propanone from 90% to 95% by skillful choice of the reaction parameters. Enzymatic synthesis with BFD are performed best in a continuously operated enzyme membrane reactor. Thus, we have established a new enzyme tool comprising a vast applicability for stereoselective synthesis.

The Nucleotide-Binding Oligomerization Domain-Like Receptor NLRC5 Is Involved in IFN-Dependent Antiviral Immune Responses

Nucleotide-binding oligomerization domain-like receptors (NLRs) are a group of intracellular proteins that mediate recognition of pathogen-associated molecular patterns or other cytosolic danger signals. Mutations in NLR genes have been linked to a variety of inflammatory diseases, underscoring their pivotal role in host defense and immunity. This report describes the genomic organization and regulation of the human NLR family member NLRC5 and aspects of cellular function of the encoded protein. We have analyzed the tissue-specific expression of NLRC5 and have characterized regulatory elements in the NLRC5 promoter region that are responsive to IFN-γ. We show that NLRC5 is upregulated in human fibroblasts postinfection with CMV and demonstrate the role of a JAK/STAT-mediated autocrine signaling loop involving IFN-γ. We demonstrate that overexpression and enforced oligomerization of NLRC5 protein results in activation of the IFN-responsive regulatory promoter elements IFN-γ activation sequence and IFN-specific response element and upregulation of antiviral target genes (e.g., IFN-α, OAS1, and PRKRIR). Finally, we demonstrate the effect of small interfering RNA-mediated knockdown of NLRC5 on a target gene level in the context of viral infection. We conclude that NLRC5 may represent a molecular switch of IFN-γ activation sequence/IFN-specific response element signaling pathways contributing to antiviral defense mechanisms.

Inhibition of hepatic fibrogenesis by matrix metalloproteinase-9 mutants in mice

Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) plays a crucial role in the pathogenesis of hepatic fibrosis and thus may represent an important therapeutic target in the design of anti‐fibrotic strategies for chronic liver disease. We present an innovative therapy based on the assignment of inactivated enzymes acting as scavengers for TIMP‐1. Hepatic fibrosis was induced in BALB/c mice by repetitive intraperitoneal CCl4 injection. The animals were treated with proteolytic inactive matrix metalloproteinase‐9 mutants (E402Q, H401A, E402H/H411E) using adenovirus‐mediated gene transfer. Application of these MMP‐9 mutants inhibited fibrogenesis, which was indicated by decreasing portal and periportal accumulation of collagen. Total hydroxyproline of liver tissue, the morphometric stage of fibrosis as well as mRNA expression of marker proteins for hepatic fibrosis in livers of E402Qand H401A‐treated mice were significantly reduced. MMP‐9 mutants suppressed transdifferentiation of hepatic stellate cells to the myofibroblast like phenotype in vitro and in vivo. Moreover, adenoviral application of the mutants MMP‐9‐H401A and ‐E402Q led to increased apoptosis of activated hepatic stellate cells, thought to be the main promoters of hepatic fibrosis. Application of MMP‐9 mutants as TIMP‐1 scavengers may provide a new therapeutic strategy for hepatic fibrosis.—Roderfeld, M., Weiskirchen, R., Wagner, S., Berres, M.‐L., Henkel, C., Grötzinger, J., Gressner, A. M., Matern, S., Roeb, E. Inhibition of hepatic fibrogenesis by matrix metalloproteinase‐9 mutants in mice. FASEB J. 20, 444–454 (2006)

The family of the IL-6-type cytokines: specificity and promiscuity of the receptor complexes.

The cytokines IL‐6, LIF, CNTF, OSM, IL‐11, and CT‐1 have been grouped into the family of IL‐6‐type cytokines, since they all require gp130 for signal transduction. Interestingly, gp130 binds directly to OSM, whereas complex formation with the other cytokines depends on additional receptor subunits. Only limited structural information on these cytokines and their receptors is available. X‐ray structures have been solved for the cytokines LIF and CNTF, whose up‐up‐down‐down four‐helix bundle is common to all of these cytokines, and for the receptors of hGH and prolactin, which contain two domains with a fibronectin III‐like fold. Since cocrystallization and x‐ray analysis of the up to four different proteins forming the receptor complexes of the IL‐6‐type cytokines is unlikely to be achieved in the near future, model building based on the existing structural information is the only approach for the time being. Here we present model structures of the complexes of human and murine IL‐6 with their receptors. Their validity can be deduced from the fact that published mutagenesis data and the different receptor specificity of human and murine IL‐6 can be understood. It is now possible to predict the relative positions and contacts for all molecules in their respective complexes. Such information can be used for the rational design of cytokine and receptor antagonists, which may have a valuable therapeutic perspective. Proteins 27:96–109