The solution structure of the kallikrein-related peptidases inhibitor SPINK6.

Kallikrein-related peptidases (KLKs) are crucial for epidermal barrier function and are involved in the proteolytic regulation of the desquamation process. Elevated KLK levels were reported in atopic dermatitis. In skin, the proteolytic activity of KLKs is regulated by specific inhibitors of the serine protease inhibitor of Kazal-type (SPINK) family. SPINK6 was shown to be expressed in human stratum corneum and is able to inhibit several KLKs such as KLK4, -5, -12, -13 and -14. In order to understand the structural traits of the specific inhibition we solved the structure of SPINK6 in solution by NMR-spectroscopy and studied its interaction with KLKs. Thereby, beside the conserved binding mode, we identified an alternate binding mode which has so far not been observed for SPINK inhibitors.

An interleukin-6 receptor-dependent molecular switch mediates signal transduction of the IL-27 cytokine subunit p28 (IL-30) via a gp130 protein receptor homodimer.

IL-27 consists of the cytokine subunit p28 and the non-signaling α-receptor EBI3. p28 was shown to additionally act via the non-signaling membrane-bound IL-6 α-receptor (IL-6R) as an agonistic cytokine but also as a gp130 ß-receptor antagonist, leading to inhibition of IL-6 signaling. Here, we developed a strategy for bacterial expression, purification, and refolding of murine p28. We show that p28 did not interfere with IL-6- or IL-27-induced signaling, indicating that p28 has no antagonistic properties. Moreover, we demonstrate that murine p28 acts as an agonistic cytokine via the murine and human IL-6R, indicating that p28 exhibits no species specificity. p28 was able to induce p28-trans-signaling via the soluble IL-6R (sIL-6R), a characteristic property that was initially described for trans-signaling of IL-6 via the sIL-6R. Of notice, p28/sIL-6R trans-signaling was inhibited by the IL-6 trans-signaling antagonist, soluble gp130. At higher concentrations, p28 but not IL-6 was able to induce signaling even in the absence of IL-6R or EBI3. Although IL-27 signals via a heterodimer of the ß-receptor chains gp130 and Wsx-1, p28/IL-6R specifically recruits two gp130 ß-receptor chains for signal transduction. The binding of p28 to a gp130/Wsx-1 heterodimer or a gp130 homodimer is highly selective and controlled by a novel molecular switch induced by EBI3 or IL-6R, respectively.

Identification of structural traits that increase the antimicrobial activity of a chimeric peptide of human ß-defensins 2 and 3.

Antimicrobial peptides participate in the first line of defence of many organisms against pathogens. In humans, the family of ß-defensins plays a pivotal role in innate immunity. Two human ß-defensins, ß-defensin-2 and -3 (HBD2 and HBD3), show substantial sequence identity and structural similarity. However, HBD3 kills Staphylococcus (S.) aureus with a 4- to 8-fold higher efficiency compared to HBD2, whereas their activities against Escherichia (E.) coli are very similar. The generation of six HBD2/HBD3-chimeric molecules led to the identification of distinct molecular regions which mediate their divergent killing properties. One of the chimeras (chimera C3) killed both E. coli and S. aureus with an even higher efficacy compared to the wild-type molecules. Due to the broad spectrum of its antimicrobial activity against many human multidrug-resistant pathogens, this HBD2/HBD3-chimeric peptide represents a promising candidate for a new class of antibiotics. In order to investigate the structural basis of its exceptional antimicrobial activity, the peptide's tertiary structure was determined by NMR spectroscopy, which allowed its direct comparison to the published structures of HBD2 and HBD3 and the identification of the activity-increasing molecular features.

Human beta-defensin 2 and beta-defensin 3 chimeric peptides reveal the structural basis of the pathogen specificity of their parent molecules.

Despite partial sequence identity and structural similarity, human ß-defensin 3 (HBD3) kills Staphylococcus aureus with a 4- to 8-fold higher efficiency than human ß-defensin 2 (HBD2), whereas the activities against Escherichia coli are identical. The design and characterization of HBD2/HBD3 chimeric peptides revealed that distinct molecular regions are responsible for their divergent killing properties. Two of the chimeras killed both E. coli and S. aureus with an even higher efficacy than the wild-type molecules. Moreover, one of these two chimeras maintained its high killing activities in the presence of physiologic salt concentrations. Due to the broad spectrum of their antimicrobial activities against many human multidrug-resistant pathogens, these two designer peptides of human origin represent promising templates for a new class of antibiotics.

A role for IL-27p28 as an antagonist of gp130-mediated signaling.

The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.

Activity of the novel peptide arminin against multiresistant human pathogens shows the considerable potential of phylogenetically ancient organisms as drug sources.

The emergence of multidrug-resistant bacteria highlights the need for new antibacterial agents. Arminin 1a is a novel antimicrobial peptide discovered during investigations of the epithelial defense of the ancient metazoan Hydra. Following proteolytic processing, the 31-amino-acid-long positively charged C-terminal part of arminin 1a exhibits potent and broad-spectrum activity against bacteria, including multiresistant human pathogenic strains, such as methicillin-resistant Staphylococcus aureus (MRSA) strains (minimal bactericidal concentration, 0.4 microM to 0.8 microM). Ultrastructural observations indicate that bacteria are killed by disruption of the bacterial cell wall. Remarkably, the antibacterial activity of arminin 1a is not affected under the physiological salt conditions of human blood. In addition, arminin 1a is a selective antibacterial agent that does not affect human erythrocyte membranes. Arminin 1a shows no sequence homology to any known antimicrobial peptide. Because of its high level of activity against multiresistant bacterial strains pathogenic for humans, the peptide arminin 1a is a promising template for a new class of antibiotics. Our data suggest that ancient metazoan organisms such as Hydra hold promise for the detection of novel antimicrobial molecules and the treatment of infections caused by multiresistant bacteria.