Effect of the combined application of heat treatment and proteases on protein stability and volatile composition of Greek white wines

Aim: This work evaluates the effects of the combined use of heat treatment (HT, 75 °C, 2 min) and proteases (P) on the protein stability and volatile composition of two white wines, obtained from the Greek cv. Assyrtiko and Moschofilero.Methods and results: Wine protein stabilization was assessed by heat test, using RP-HPLC determination of pathogenesis-related proteins (PRP) and by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The impact of bentonite and P + HT treatment on wine aroma profile was evaluated by GC-MS with liquid–liquid extraction. According to the heat test, in Assyrtiko wine the level of stability achieved with P + HT was comparable with that obtained by bentonite fining and for Moschofilero wine – where protein instability was higher – bentonite was more efficient. RP-HPLC profiles showed that, in general, higher percentages of chitinases (CH) than thaumatin-like proteins (TLP) were removed by both bentonite and P + HT, with a similar efficiency for the two treatments and sometimes better performances for the latter. Conversely, TLP were removed more efficiently by bentonite, even if some fractions were eliminated to a slightly higher extent by proteases. In the conditions of the experiment, bentonite resulted in minor changes to the wine aroma profile. However, heating during protease treatment modified wine volatile composition, reducing the concentration of esters produced during fermentation while simultaneously increasing the contents of certain esters characteristic of aging such as ethyl lactate.Conclusions: The combination of proteases and heat treatment may be a promising technique for protein stabilization of wines. However, further investigations are needed to optimize the time:temperature ratio of the heat treatment in order to obtain the maximum protease activity and the minimum thermal deterioration of the wine quality.Significance and impact of the study: The results of this study have a practical interest for both the scientific community and wine sector, contributing to knowledge of the efficacy and limitations of the use of protease enzymes for wine stabilization

The Interaction of the Chemotherapeutic Drug Chlorambucil with Human Glutathione Transferase A1-1: Kinetic and Structural Analysis

Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/ CBL-GSH complex was solved at 2.1 A° resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the Cterminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated

Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3

Vascular endothelial growth factor C (VEGF-C) is a key mediator of lymphangiogenesis, acting via its receptors VEGF-R2 and VEGF-R3. High expression of VEGF-C in tumors correlates with increased lymphatic vessel density, lymphatic vessel invasion, sentinel lymph node metastasis and poor prognosis. Recently, we found that in a chemically induced skin carcinoma model, increased VEGF-C drainage from the tumor enhanced lymphangiogenesis in the sentinel lymph node and facilitated metastatic spread of cancer cells via the lymphatics. Hence, interference with the VEGF-C/VEGF-R3 axis holds promise to block metastatic spread, as recently shown by use of a neutralizing anti-VEGF-R3 antibody and a soluble VEGF-R3 (VEGF-C/D trap). By antibody phage-display, we have developed a human monoclonal antibody fragment (single-chain Fragment variable, scFv) that binds with high specificity and affinity to the fully processed mature form of human VEGF-C. The scFv binds to an epitope on VEGF-C that is important for receptor binding, since binding of the scFv to VEGF-C dose-dependently inhibits the binding of VEGF-C to VEGF-R2 and VEGF-R3 as shown by BIAcore and ELISA analyses. Interestingly, the variable heavy domain (VH) of the anti-VEGF-C scFv, which contains a mutation typical for camelid heavy chain-only antibodies, is sufficient for binding VEGF-C. This reduced the size of the potentially VEGF-C-blocking antibody fragment to only 14.6 kDa. Anti-VEGF-C VH-based immunoproteins hold promise to block the lymphangiogenic activity of VEGF-C, which would present a significant advance in inhibiting lymphatic-based metastatic spread of certain cancer types

High-resolution crystal structures of a “half sandwich”-type Ru(II) coordination compound bound to hen egg-white lysozyme and proteinase K

The high-resolution X-ray crystal structures of the adducts formed between the “half sandwich”-type Ru(II) coordination compound [Ru$^{II}$(1,4,7-trithiacyclononane)(ethane-1,2-diamine)Cl]$^+$ and two proteins, namely hen egg-white lysozyme and proteinase K, are presented. The structures unveil that upon reaction with both enzymes the Ru(II) compound is coordinated by solvent-exposed aspartate residues after releasing the chloride ligand (Asp101 in lysozyme, Asp200 and Asp260 in proteinase K), while retaining the two chelating ligands. The adduct with Asp101 residue at the catalytic cleft of lysozyme is accompanied by residue-specific conformational changes to accommodate the Ru(II) fragment, whereas the complexes bound at the two calcium-binding sites of proteinase K revealed minimal structural perturbation of the enzyme. To the best of our knowledge, proteinase K is used here for the first time as a model system of protein metalation and these are the first X-ray crystal structures of protein adducts of a Ru(II) coordination compound that maintains its coordination sphere almost intact upon binding. Our data demonstrate the role of ligands in stabilizing the protein adducts via hydrophobic/aromatic or hydrogen-bonding interactions, as well as their underlying role in the selection of specific sites on the electrostatic potential surface of the enzymes

Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from [i]Podospora anserina[/i] and [i]Myceliophthora thermophila[/i]

Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl d-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three d-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-d-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs

Prednisolone exerts late mitogenic and biphasic effects on resistant acute lymphoblastic leukemia cells: Relation to early gene expression

Resistance or sensitivity to glucocorticoids is considered to be of crucial importance for disease prognosis in childhood acute lymphoblastic leukemia. Prednisolone exerted a delayed biphasic effect on the resistant CCRF-CEM leukemic cell line, necrotic at low doses and apoptotic at higher doses. At low doses, prednisolone exerted a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolone’s mitogenic and cell death effects were counterbalanced. Early gene microarray analysis revealed notable differences in 40 genes. The mitogenic/biphasic effects of prednisolone are of clinical importance in the case of resistant leukemic cells. This approach might lead to the identification of gene candidates for future molecular drug targets in combination therapy with glucocorticoids, along with early markers for glucocorticoid resistance. (C) 2009 Elsevier Ltd. All rights reserved

Structural representation illustrating the lock-and-key motif in hGSTA1-1 and a possible mode of communication between the two subunits.

<p>The key residues Met51, Phe52, Cys112 and the GSH–CBL adducts are shown in licorice representation.</p

Crystal structure of hGSTA1-1/GSH–CBL complex.

<p>(A) Ribbon representation of monomer A with the GSH–CBL adduct atoms shown as van der Waals spheres and coloured orange for C, blue for N, red for O, yellow for S and green for Cl. (B) Representative 2Fo-Fc electron density map contoured at 1σ in the vicinity of bound GSH–CBL superimposed on refined crystal structure (left) in comparison with the simulated GSH–CBL adduct coloured according to the B-factor calculated from 10-ns molecular dynamics (right). (C) Residue-specific interactions of CBL moiety (orange licorice) in monomer A (cyan licorice). (D) Residue-specific interactions of the GSH moiety (orange licorice) from monomer A (blue ribbons) and monomer B (red ribbons).</p

Root mean square fluctuations (RMSF) of hGSTA1-1 Cα atoms calculated for the apo (black) and the holo complex with GSH–CBL (red) from 10-ns molecular dynamics simulations.

<p>The graph shows the mean RMSF value of the two monomers.</p