Structural basis for cross-reactivity and conformation fluctuation of the major beech pollen allergen Fag s 1.

Fag s 1 is a member of the Pathogen Related protein family 10 (PR-10) and can elicit cross-reaction with IgE antibodies produced against the birch pollen allergen Bet v 1. The Nuclear Magnetic Resonance (NMR) structure of Fag s 1 is presented along with its dynamic properties. It shares 66% identity with Bet v 1 and exhibits the expected three α-helices and seven ß-sheets arranged as a semi-beta barrel and exposing the residues mapped as the Bet v 1 IgE epitope. The structural dynamics of Fag s 1 were monitored on the fast and intermediate timescales, using relaxation rates. The complex dynamics of Fag s 1 are closely related to the internal cavity, and they modulate IgE and ligand binding.

Hydration and conformational equilibrium in yeast thioredoxin 1: implication for H(+) exchange.

One of the ancestral features of thioredoxins is the presence of a water cavity. Here, we report that a largely hydrated, conserved, buried aspartic acid in the water cavity modulates the dynamics of the interacting loops of yeast thioredoxin 1 (yTrx1). It is well-established that the aspartic acid, Asp24 for yTrx1, works as a proton acceptor in the reduction of the target protein. We propose a complementary role for Asp24 of coupling hydration and conformational motion of the water cavity and interacting loops. The intimate contact between the water cavity and the interacting loops means that motion at the water cavity will affect the interacting loops and vice versa. The D24N mutation alters the conformational equilibrium for both the oxidized and reduced states, quenching the conformational motion in the water cavity. By measuring the hydration and molecular dynamics simulation of wild-type yTrx1 and the D24N mutant, we showed that Asn24 is more exposed to water than Asp24 and the water cavity is smaller in the mutant, closing the inner part of the water cavity. We discuss how the conformational equilibrium contributes to the mechanism of catalysis and H(+) exchange.

Mapping the interactions between a major pollen allergen and human IgE antibodies.

The interaction of specific IgE antibodies with allergens is a key event in the induction of allergic symptoms, thus representing an important target for therapeutic interventions in Type I allergies. We report here the solution NMR structure of Art v 1, the major mugwort pollen allergen. Art v 1 is the first protein structure with an allergenic defensin fold linked to a polyproline domain, which has not been identified in any reported allergen structure in the PDB. Moreover, the direct interaction of polyclonal IgE antibodies from an allergic patient has been mapped on the surface of an allergen for the first time. The data presented herein provide the basis for the design of tools for safe and effective vaccination against mugwort pollen allergy.

Sequence-specific 1H, 15N and 13C resonance assignments of Art v 1: a proline-rich allergen of Artemisia vulgaris pollen.

Art v 1 is the major allergen of Artemisia vulgaris. The IgE raised against Art v 1 not only can cross-react with other proteins from the Asteraceae family members but also with components of various forms of food. Art v 1 is an important target for immunotherapy strategies, including vaccination with hypoallergenic derivatives or chimeras. We report the (1)H, (13)C, and (15)N resonance assignments of the recombinant Art v 1 and identification of secondary structures based on (13)C chemical shifts.

Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate.

3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.