Pharmacological Targets of Kaempferol Within Inflammatory Pathways-A Hint Towards the Central Role of Tryptophan Metabolism.

The flavonoid kaempferol is almost ubiquitously contained in edible and medicinal plants and exerts a broad range of interesting pharmacological activities. Interactions with central inflammatory processes can be exploited to treat or attenuate symptoms of disorders associated with chronic immune activation during infections, malignancies, and neurodegenerative or cardiovascular disorders. Many drugs, phytochemicals, and nutritional components target the catabolism of the essential amino acid tryptophan by indoleamine 2,3-dioxygenase 1 (IDO-1) for immunomodulation. We studied the effects of kaempferol by in vitro models with human peripheral blood mononuclear cells (PBMC) and THP-1 derived human myelomonocytic cell lines. Kaempferol suppressed interferon-γ dependent immunometabolic pathways: Formation of the oxidative stress biomarker neopterin and catabolism of tryptophan were inhibited dose-dependently in stimulated cells. In-silico docking studies revealed a potential interaction of kaempferol with the catalytic domain of IDO-1. Kaempferol stimulated nuclear factor kappa B (NF-κB) signaling in lipopolysaccharide (LPS)-treated THP-1 cells, thereby increasing the mRNA expression of interleukin (IL) 1 beta, tumor necrosis factor, and nuclear factor kappa B subunit 1, while IL6 was downregulated. Data suggest that concerted effects of kaempferol on multiple immunologically relevant targets are responsible for its immunomodulatory activity. However, the immunosuppressive effects may be more relevant in a T-cell dominated context.

Protease Inhibitors in View of Peptide Substrate Databases.

Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods.

Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.

BACKGROUND: The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties. OBJECTIVE: We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1. METHODS: By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model. RESULTS: Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment. CONCLUSIONS: Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.

Inhibition of Collagenase by Mycosporine-like Amino Acids from Marine Sources.

Matrix metalloproteinases play an important role in extracellular matrix remodeling. Excessive activity of these enzymes can be induced by UV light and leads to skin damage, a process known as photoaging. In this study, we investigated the collagenase inhibition potential of mycosporine-like amino acids, compounds that have been isolated from marine organisms and are known photoprotectants against UV-A and UV-B. For this purpose, the commonly used collagenase assay was optimized and for the first time validated in terms of relationships between enzyme-substrate concentrations, temperature, incubation time, and enzyme stability. Three compounds were isolated from the marine red algae Porphyra sp. and Palmaria palmata, and evaluated for their inhibitory properties against Chlostridium histolyticum collagenase. A dose-dependent, but very moderate, inhibition was observed for all substances and IC50 values of 104.0 µM for shinorine, 105.9 µM for porphyra, and 158.9 µM for palythine were determined. Additionally, computer-aided docking models suggested that the mycosporine-like amino acids binding to the active site of the enzyme is a competitive inhibition.

Ligand binding modulates the structural dynamics and compactness of the major birch pollen allergen.

Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.

The impact of nitration on the structure and immunogenicity of the major birch pollen allergen Bet v 1.0101.

Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of TH1-priming cytokines, thus favoring a TH2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.