Structures of an active-site mutant of a plant 1,3-ß-glucanase in complex with oligosaccharide products of hydrolysis.

Plant endo-1,3-ß-glucanases are involved in important physiological processes such as defence mechanisms, cell division and flowering. They hydrolyze (1→3)-ß-glucans, with very limited activity towards mixed (1→3,1→4)-ß-glucans and branched (1→3,1→6)-ß-glucans. Here, crystal structures of the potato (Solanum tuberosum) endo-1,3-ß-glucanase GLUB20-2 with the nucleophilic Glu259 residue substituted by alanine (E259A) are reported. Despite this active-site mutation, the protein retained residual endoglucanase activity and when incubated in the crystallization buffer with a linear hexameric substrate derived from (1→3)-ß-glucan (laminarahexose) cleaved it in two different ways, generating trisaccharides and tetrasaccharides, as confirmed by mass spectrometry. The trisaccharide (laminaratriose) shows higher binding affinity and was found to fully occupy the -1, -2 and -3 sites of the active-site cleft, even at a low molar excess of the substrate. At elevated substrate concentration the tetrasaccharide molecule (laminaratetrose) also occupies the active site, spanning the opposite sites +1, +2, +3 and +4 of the cleft. These are the first crystal structures of a plant glycoside hydrolase family 17 (GH17) member to reveal the protein-saccharide interactions and were determined at resolutions of 1.68 and 1.55 Å, respectively. The geometry of the active-site cleft clearly precludes any (1→4)-ß-glucan topology at the subsites from -3 to +4 and could possibly accommodate ß-1,6-branching only at subsites +1 and +2. The glucose units at subsites -1 and -2 interact with highly conserved protein residues. In contrast, subsites -3, +3 and +4 are variable, suggesting that the mode of glucose binding at these sites may vary between different plant endo-1,3-ß-glucanases. Low substrate affinity is observed at subsites +1 and +2, as manifested by disorder of the glycosyl units there.

Two high-resolution structures of potato endo-1,3-ß-glucanase reveal subdomain flexibility with implications for substrate binding.

Endo-1,3-ß-glucanases are widely distributed among bacteria, fungi and higher plants. They are responsible for hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubstituted ß-1,3-linked glucosyl residues. The plant enzymes belong to glycoside hydrolase family 17 (GH17) and are also members of class 2 of pathogenesis-related (PR) proteins. X-ray diffraction data were collected to 1.40 and 1.26 Å resolution from two crystals of endo-1,3-ß-glucanase from Solanum tuberosum (potato, cultivar Désirée) which, despite having a similar packing framework, represented two separate crystal forms. In particular, they differed in the Matthews coefficient and are consequently referred to as higher density (HD; 1.40 Å resolution) and lower density (LD; 1.26 Å resolution) forms. The general fold of the protein resembles that of other known plant endo-1,3-ß-glucanases and is defined by a (ß/α)(8)-barrel with an additional subdomain built around the C-terminal half of the barrel. The structures revealed high flexibility of the subdomain, which forms part of the catalytic cleft. Comparison with structures of other GH17 endo-1,3-ß-glucanases revealed differences in the arrangement of the secondary-structure elements in this region, which can be correlated with sequence variability and may suggest distinct substrate-binding patterns. The crystal structures revealed an unusual packing mode, clearly visible in the LD structure, caused by the presence of the C-terminal His(6) tag, which extends from the compact fold of the enzyme molecule and docks in the catalytic cleft of a neighbouring molecule. In this way, an infinite chain of His-tag-linked protein molecules is formed along the c direction.

[The investigations of the role of toll-like receptors (TLR) in host response to parasitic infection on the current background regarding TLR in mammals and the model nematode Caenorhabditis elegans]. / Badania roli receptorów Toll-podobnych (TLR) w odpowiedzi zywiciela na inwazje pasozytnicza na tle wspólczesnej wiedzy o TLR u ssaków i modelowego nicienia Caenorhabditis elegans.

Toll-like receptors (TLRs) are amongst the most highly conserved in the evolution of receptor family, being found in both immune and other cells. TLRs were observed in vascular endothelial cells, epithelial cells, microglia cells, adipocytes, and intestinal and renal cells. TLRs plays a key role in the innate immune response to a variety of pathogens. At present, very little is known about the role of TLRs in host defense against parasitic pathogen infections. The first study shows that TLRs contribute to both innate and adaptive immune responses following infection with protozoan parasite Leishmania major. The TLRs recognizing PAMPs associated with the parasite L. major are essential for the activation of the innate and adaptive immune responses to infection. A study concerning recognition of the role of TLRs in the host-parasite relationship would be an interesting challenge for future study.

[Effect in vitro of albendazole on the kinetics of cytosolic glutathione transferase from the rat liver]. / Badania in vitro wplywu albendazolu na kinetyke reakcji ka- talizowanej przez cytozolowa transferaze glutationowa (GST) z watroby szczura.

INTRODUCTION: Since the idea of multifunctional mode of action of anthelmintics is considered and in experimental trichinellosis in vivo albendazole seems to act as an allosteric activator of cytosolic GST from mice muscles, in this study a termosensitivity after in vitro incubation with albendazole of purified commercial cytosolic glutathione transferase (GST) from the rat liver was investigated. METHODS: Two extremal temperatures: -80 degrees C and +30 degrees C were used to destroy the dimer in quaternary structure of this enzyme. RESULTS: In control preparations both extremal temperatures destroy this structure, so the Michaelis-Menten kinetic curves of substrate saturation show the typical hyperbolic shape. After a long (15 h) freezing at -80 degrees C or heating (up to 14 h at +30 degrees C) the kinetics of substrate saturation of GST after incubation with albendazole show the sigmoidal or "double sigmoidal" shape, pointing out the quaternary GST structure as a complex of "frozen subunits". Drug inhibits about 6-times the total activity of GST after incubation at +30 degrees C. We conclude that albendazole in vitro influences the structure of cytosolic GST from the rat liver and inhibits its activity, but, in opposite to in vivo study in mouse muscles infected with Trichinella spiralis larvae, does not act as an activator of this enzyme.

Evaluation of effects of albendazole on the kinetics of cytosolic glutathione transferase in skeletal muscles during experimental trichinellosis in mice.

In this study, the effect of trichinellosis as well as the effect of albendazole treatment on the kinetics of substrate saturation of cytosolic glutathione transferase (GST) in the skeletal muscles from infected mice was examined because the idea of multifunctional mode of action of anthelmintic drugs was considered. Our results pointed out to the influence of trichinellosis and albendazole treatment on the quaternary structure of GST in mouse muscles. A double reciprocal plot of GST saturation in control mice was biphasic with apparent low and high K (m) values equal to 0.54 and 1.0 mM, respectively. Infection with Trichinella spiralis in the third week postinfection caused a 2.3-fold increase in the high K (m) value and at the same time a 2.8-fold decrease in the low K (m). In the sixth week postinfection, the high K (m) value was unchanged, but the low K (m) value increased 2.3 times. Calculated from the double reciprocal plot of GST substrate saturation in muscles from infected and treated mice (measured in the third week postinfection only), the high K (m) value increased 1.4 times relative to the respective controls. The normal substrate saturation plot of GST in treated mice has a clearly "double sigmoid" character. Our results suggest that despite the complicated character of the GST saturation curve, albendazole seems to act as an allosteric activator for cytosolic GST in infected mouse muscles.

[The intriguing influence of albendazole on glutathione reductase activity in the muscles from Trichinella spiralis infected mice]. / Intrygujacy wplyw albendazolu na aktywnosc reduktazy glutationu w miesniach myszy zarazonych Trichinella spiralis.

A clear stimulation of the activity of glutathione reductase (RG, EC 1.6.4.2), one of the major enzymes participating in glutathione metabolism, was observed in the skeletal muscles from Trichinella spiralis infected mice. The maximal, almost two-fold increase in RG activity was observed in week 5 post-infection. Administration of albendazole, an anthelmintic widely used in trichinellosis treatment, resulted in an additional, about 40% stimulation of RG activity (small, but statistically significant) on week 2 post-infection. In weeks 6 and 7 post-infection, when no stimulation of RG activity in infected mice caused by drug was observed, the drug modified the structure of that allosteric enzyme, affecting the kinetics of its substrate saturation in this way that the shape of the substrate saturation curve changed from "double sigmoidal", typical of this enzyme kinetics in the muscles from infected mice, to hyperbolic, typical of this enzyme kinetics in the control, uninfected animals.

RNAi and viral vectors as useful tools in the functional genomics of plants. Construction of BMV-based vectors for RNA delivery into plant cells.

The sequencing of several complete genomes and the development of a DNA microarray technology are among the most important achievements of molecular biology. They gave the proper grounds for the development of modern functional genomics. However, there is one additional condition which needs to be satisfied to truely enable the study of how a genome works: a suitable method of selectively inducing and silencing the expression of each individual gene. The methods used so far have usually only permitted the influencing of gene expression through genetic manipulations at the DNA level (genetically modified plants). The discovery of RNA interference (RNAi) opens up completely new possibilities of research on the functioning of particular plant genes, without the necessity of altering the genome structure. In this case, interference takes place at the transcript level. Thus, at any given moment during plant development, the expression of a specific gene (or several genes) can be inhibited, even if it is important for the survival of the organism under study. To this end, a double-stranded RNA inducing the RNAi phenomenon has to be delivered into the plant cell. Here we describe the construction of four brome mosaic virus-based vectors, which, as our preliminary data indicate, can be used to transfer RNA into barley cells.