Recent advances in protein engineering and biotechnological applications of glutathione transferases

Glutathione transferases (GSTs, EC 2.5.1.18) are a widespread family of enzymes that play a central role in the detoxification, metabolism, and transport or sequestration of endogenous or xenobiotic compounds. During the last two decades, delineation of the important structural and catalytic features of GSTs has laid the groundwork for engineering GSTs, involving both rational and random approaches, aiming to create new variants with new or altered properties. These approaches have expanded the usefulness of native GSTs, not only for understanding the fundamentals of molecular detoxification mechanisms, but also for the development medical, analytical, environmental, and agricultural applications. This review article attempts to summarize successful examples and current developments on GST engineering, highlighting in parallel the recent knowledge gained on their phylogenetic relationships, structural/catalytic features, and biotechnological applications.     

Water relations of yellow sweetclover under the synergy of drought and selenium addition

In the last two decades drought and elevated toxic metal concentration phenomena in plants have gained the interest of the scientific world. Nevertheless, up to day relatively little ecophysiological research concerning the effect of water stress and elevated selenium (Se) concentration on plant water relations is available. A pot experiment was conducted in order to evaluate the effects of the implied synergy of drought and Se uptake on water relations of yellow sweetclover (Melilotus officinalis L.). The effects of two different Se concentrations (0 mg Se L^?1 irrigation water, 3 mg Se L^?1 irrigation water) and two water regimes (full irrigation — limited irrigation) applied to seedlings of yellow sweetclover were detected by measuring changes in water potential, relative water content, stomatal conductance, transpiration rate and tissue Se concentration. The findings suggest that yellow sweetclover, concentrating up to 200 μg Se g^?1 dry weight in its tissues, could be considered a secondary Se accumulator. Se effect on water relations was more evident under limited irrigation, as expressed by decreased values of leaf water potential, transpiration rate and stomatal conductance, limiting the flux rate of the water solution in the conducting system of the plant.     

Wild blueberry (Vaccinium angustifolium) consumption affects the composition and structure of glycosaminoglycans in Sprague-Dawley rat aorta

It has been documented that increased intake of polyphenols may provide protection against coronary heart disease and stroke. Blueberries (Vaccinium angustifolium) are one of the richest sources of antioxidants among fruits and vegetables. Phenolic compounds from berry extracts inhibit human low density lipoprotein and liposome oxidation. Glycosaminoglycans (GAGs) and proteoglycans (PGs) are structural components of aortas with great structural diversity. Their interaction with compounds such as enzymes, cytokines, growth factors, proteins and lipoproteins and their subsequent role in degenerative diseases has been documented. We investigated the effects of a diet rich in blueberries on the content and structure of GAGs. Sprague-Dawley rats were fed either a control (C) or a blueberry (B) diet for 13 weeks. Aortic tissue GAGs were isolated with papain digestion, alkaline borohydride treatment and anion-exchange chromatography. Cellulose acetate electrophoresis and treatment of the fractions with specific lyases revealed the presence of three GAG populations, i.e. hyaluronan (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). Disaccharide composition was determined by high-performance capillary electrophoresis following enzymatic degradation. A 13% higher amount of total GAGs in aortas of B-fed rats was attributed to a higher content of GalAGs (67%). Determination of the sulfated disaccharides showed an overall lower concentration of oversulfated disaccharides in both HS and GalAG populations in the aortas of the B group. Our results demonstrate for the first time that a diet rich in blueberries results in structural alterations in rat aortic tissue GAGs. These changes may affect cellular signal transduction pathways and could have major consequences for the biological function of GAG molecules within the vascular environment.     

Dietary manganese affects the concentration, composition and sulfation pattern of heparan sulfate glycosaminoglycans in Sprague-Dawley rat aorta

We examined the effect of dietary Mn on the composition and structure of heparan sulfate (HS) glycosaminoglycans (GAGs) of rat aorta. Animals were randomly assigned to either a Mn deficient (MnD), adequate (MnA) or supplemented (MnS) diet (Mn<1, 10–15 and 45–50 ppm, respectively). After 15 weeks, aortic tissue GAGs were isolated with papain digestion, alkaline borohydride treatment and anion-exchange chromatography. Cellulose acetate electrophoresis and treatment of the fractions with specific lyases revealed the presence of three GAG populations, i.e. hyaluronan (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). Disaccharide composition of the HS fractions was determined by HPCE following treatment with heparin lyases I, II and III. In MnS aortas we observed increased concentration of total GalAGs and decreased concentration of HS and HA, when compared to MnA aortas. Aortas from MnD and MnA rats appeared to have similar distribution of individual GAGs. Heparan sulfate chains of MnS aortas contained higher (41%) concentration of non-sulfated units compared to MnA ones. Variable amounts of trisulfated and disulfated units were found only in MnD and MnA groups but not in MnS. Our results demonstrate that HS biosynthesis in the rat aorta undergoes marked structural modifications that depend upon dietary Mn intake. The reduced expression and undersulfation of HSPGs with Mn supplementation might indicate a reduced ability of vascular cells to interact with biologically active molecules such as growth factors. Alterations in cell-membrane binding ability to a variety of extracellular ligands might affect signal-transduction pathways and arterial functional properties.     

Evolutionary relationships within European Monochamus (Coleoptera: Cerambycidae) highlight the role of altitude in species delineation

Phylogenetic relationships within the European Monochamus (Coleoptera: Cerambycidae) remain understudied despite their increasing importance in the Pine Wood Nematode spread in Europe. To clarify the delimitation and the evolutionary history of the two main European Monochamus species, Monochamus galloprovincialis and Monochamus sutor, as well as their sub‐species, a comparative study using morphological, molecular, and biogeographical criterions was conducted. Four morphological characters, including a newly‐described morphological character on the male genitalia, separated the two species. Additionally, molecular data revealed twelve and two single nucleotide polymorphisms in cytochrome oxidase c subunit I and 28S, respectively, supporting species segregation. By contrast, incongruence between morphological and genetic results did not allow discriminating the sub‐species of M. galloprovincialis and M. sutor, even though mitochondrial DNA revealed intraspecific differentiation, mostly consenting to a multiple refugia origin. Within‐species variability was explained to a large extent by biogeography (i.e. altitude, climate). These different ecological adaptations within beetle species, together with potential climate change impact, increase the risk of spreading the nematode across Europe to novel conifer hosts and challenge the European biosecurity. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 354–376.     

Wild blueberry (V. angustifolium)-enriched diets alter aortic glycosaminoglycan profile in the spontaneously hypertensive rat

Glycosaminoglycans (GAGs) are essential polysaccharide components of extracellular matrix and cell surface with key roles on numerous vascular wall functions. Previous studies have documented a role of wild blueberries on the GAG profile of the Sprague-Dawley rat with a functional endothelium as well as in the vascular tone of the spontaneously hypertensive rat (SHR) with endothelial dysfunction. In the present study, the effect of wild blueberries on the composition and structure of aortic GAGs was examined in 20-week-old SHRs after 8 weeks on a control (C) or a wild blueberry-enriched diet (WB). Aortic tissue GAGs were isolated following pronase digestion and anion-exchange chromatography. Treatment of the isolated populations with specific GAG-degrading lyases and subsequent electrophoretic profiling revealed the presence of three GAG species, i.e., hyaluronic acid (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). A notable reduction of the total sulfated GAGs and a redistribution of the aortic GAG pattern were recorded in the WB as compared to the C group: a 25% and 10% increase in HA and HS, respectively, and an 11% decrease in GalAGs. Fine biochemical analysis of GalAGs at the level of constituent disaccharides with high-performance capillary electrophoresis revealed a notable increase of nonsulfated (18.0% vs. 10.7%) and a decrease of disulfated disaccharides (2.2% vs. 5.3%) in the WB aorta. This is the first study to report the redistribution of GAGs at the level of composition and their fine structural characteristics with implications for the endothelial dysfunction of the SHR.     

Enhanced neuronal plasticity and elevated endogenous sAPPα levels in mice over-expressing MMP9

Evidence accumulating during the past few years points to a significant role of matrix metalloproteinase 9 (MMP9) enzymatic activity in synaptic plasticity and cognitive processes. We have previously demonstrated that MMP9 is involved in receptor-mediated α-secretase-like cleavage of APP in vitro, resulting in increased secretion of sAPPα, the soluble N-terminal product of the non-amyloidogenic pathway known to be involved in neuronal plasticity and memory formation. To study the in vivo role of MMP9, we have generated transgenic mice over-expressing MMP9 in the brain. Herein, we demonstrate that MMP9 transgenic animals display enhanced performance in the non-spatial novel object recognition and the spatial water-maze task and that their enhanced performance was accompanied by increased dendritic spine density in the hippocampus and cortex following behavioural testing. Consistent with the above observations, the electrophysiological analysis revealed prolonged maintenance of long-term synaptic potentiation in hippocampal slices from MMP9 transgenic mice. Moreover, elevated sAPPα levels in the hippocampus and cortex of MPP9 transgenic animals were also observed. Overall, our results extend previous findings on the physiological role of MMP9 in neuronal plasticity and furthermore reveal that, APP may be one of the physiological proteolytic targets of MMP9 in vivo.     

Investigating the entire course of telithromycin binding to Escherichia coli ribosomes

Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (K(T) = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (K(T)* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.