Investigating the role of metal ions in the catalytic mechanism of the yeast RNA triphosphatase

The Saccharomyces cerevisiae RNA triphosphatase (Cet1) requires the presence of metal ion cofactors to catalyze its phosphohydrolase activity, the first step in the formation of the 5'-terminal cap structure of mRNAs. We have used endogenous tryptophan fluorescence studies to elucidate both the nature and the role(s) of the metal ions in the Cet1-mediated phosphohydrolase reaction. The association of Mg2+, Mn2+, and Co2+ ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was then used to determine the apparent dissociation constants for these ions. Subsequent dual ligand titration experiments demonstrated that the metal ions bind to a common site, for which they compete. The kinetics of real-time metal ion binding to the Cet1 protein were also investigated, and the effects on RNA and nucleotide binding were evaluated. To provide additional insight into the relationship between Cet1 structure and metal ion binding, we correlated the effect of ion binding on protein structure using both circular dichroism and guanidium hydrochloride-induced denaturation as structural indicators. Our data indicate that binding of RNA, nucleotides, and metal ion cofactors does not lead to significant structural modifications of the Cet1 architecture. This suggests a model in which Cet1 possesses a preformed active site, and where major domain rearrangements are not required to form an active catalytic site. Finally, denaturation studies demonstrate that the metal ion cofactors can act by stabilizing the ground state binding of the phosphohydrolase substrate.     

Improving the biotreatment of hydrocarbons-contaminated soils by addition of activated sludge taken from the wastewater treatment facilities of an oil refinery

Addition of activated sludge taken from the wastewater treatment facilities ofan oil refinery to a soil contaminated with oily sludge stimulated hydrocarbonbiodegradation in microcosms, bioreactors and biopile. Microcosms containing50 g of soil to which 0.07 % (w/w) of activated sludge was added presented ahigher degradation of alkanes (80 % vs 24 %) and polycyclic aromatic hydrocarbons(PAHs) (77 % vs 49 %) as compared to the one receiving only water, after 30days of incubation at room temperature. Addition of ammonium nitrate or sterilesludge filtrate instead of activated sludge resulted in a similar removal of PAHsbut not of alkanes suggesting that the nitrogen contained in the activated sludgeplays a major role in the degradation of PAHs while microorganisms of thesludge are active against alkanes. Addition of sludge also stimulated hydrocarbonbiodegradation in 10-kg bioreactors operated during 60 days and in a 50-m³ biopile operated during 126 days. This biopile treatment allowed the use of the soil for industrial purpose based on provincial regulation (``C' criteria). In contrast, the soil of the control biopile that received only water still exceeded C criteria for C₁₀–C₅₀ hydrocarbons, total PAHs, chrysene and benzo[a]anthracene.The stimulation effect of sludge was stronger on the 4-rings than on 2-rings PAHs.The soil of the biopile that received sludge was 4–5 times less toxic than the control. These results suggest that this particular type of activated sludge could be used to increase the efficiency of the treatment of hydrocarbon-contaminated soils in a biopile.     

Purification and characterization of a 4-hydroxybenzoate decarboxylase from an anaerobic coculture

The oxygen-sensitive 4-hydroxybenzoate decarboxylase (4OHB-DC) activity from a phenol-carboxylating coculture, consisting of Clostridium-like strain 6 and an unidentified strain 7, was studied. Assays done with cell extracts showed that the optimal pH was 5.0-6.5 and the Km was 5.4 mM. The activity decreased by 50% in the presence of 5 mM EDTA, and it was restored and even enhanced by the addition of Mg++, Mn++, Zn++, or Ca++. After purification, the molecular mass of the enzyme was estimated as 420 kDa by gel chromatography, and as 119 kDa by SDS-PAGE, suggesting a homotetrameric structure. Its pI was 5.6. The N-terminal amino acid sequence showed 95% and 76% homology with the pyruvate-flavodoxin oxidoreductase (nifJ gene product) from Enterobacter agglomerans and Klebsiella pneumoniae, respectively. The purified enzyme also slowly catalyzed the reverse reaction, that is the phenol carboxylation. These characteristics suggest that this enzyme is different from other known decarboxylases. This includes the 4OHB-DC from Clostridium hydroxybenzoicum, which is the only one that had been purified before.     

The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. In this study, the substrate preference of this enzyme was investigated by mutational analysis, X-ray crystallography and homology modelling. The crystal structure of hCBG was solved by the molecular replacement method and refined at 2.7 A resolution. The main-chain fold of the enzyme belongs to the (beta/alpha)(8) barrel structure, which is common to family 1 glycoside hydrolases. The active site is located at the bottom of a pocket (about 16 A deep) formed by large surface loops, surrounding the C termini of the barrel of beta-strands. As for all the clan of GH-A enzymes, the two catalytic glutamate residues are located on strand 4 (the acid/base Glu165) and on strand 7 (the nucleophile Glu373). Although many features of hCBG were shown to be very similar to previously described enzymes from this family, crucial differences were observed in the surface loops surrounding the aglycone binding site, and these are likely to strongly influence the substrate specificity. The positioning of a substrate molecule (quercetin-4'-glucoside) by homology modelling revealed that hydrophobic interactions dominate the binding of the aglycone moiety. In particular, Val168, Trp345, Phe225, Phe179, Phe334 and Phe433 were identified as likely to be important in determining substrate specificity in hCBG, and site-directed mutagenesis supported a key role for some of these residues.     

Characterization of conserved viral leader RNA sequences that stimulate innate immunity through TLRs

Viruses of the order Mononegavirales encompass life-threatening pathogens with single-stranded segmented or nonsegmented negative-strand RNA genomes. The RNA genomes are characterized by highly conserved sequences at the extreme untranslated 3' and 5' termini that are most important for virus infection and viral RNA synthetic processes. The 3' terminal genome regions of negative-strand viruses such as vesicular stomatitis virus, Sendai virus, or influenza virus contain a high number of conserved U and G nucleotides, and synthetic oligoribonucleotides encoding such sequences stimulate sequence-dependent cytokine responses via TLR7 and TLR8. Immune cells responding to such sequences include NK cells, NK/T cells, plasmacytoid, and myeloid dendritic cells, as well as monocytes and B cells. Strong Th1 and pro-inflammatory cytokine responses are also induced upon in vivo application of oligoribonucleotides. It appears possible that the presence of highly conserved untranslated terminal regions in the viral genome fulfilling fundamental functions for the viral replication may enable the host to induce directed innate immune defense mechanisms, by allowing pathogen detection through essential RNA regions that the virus cannot readily mutate.     

Quantitative fluorometric analysis of the protective effect of chitosan on thermal unfolding of catalytically active native and genetically-engineered chitosanases

We have taken advantage of the intrinsic fluorescence properties of chitosanases to rapidly and quantitatively evaluate the protective effect of chitosan against thermal denaturation of chitosanases. The studies were done using wild type chitosanases N174 produced by Streptomyces sp. N174 and SCO produced by Streptomyces coelicolor A3(2). In addition, two mutants of N174 genetically engineered by single amino acid substitutions (A104L and K164R) and one "consensus" (N174-CONS) chitosanase designed by multiple amino acid substitutions of N174 were analyzed. Chitosan used had a weight average molecular weight (Mw) of 220 kDa and was 85% deacetylated. Results showed a pH and concentration-dependent protective effect of chitosan in all the cases. However, the extent of thermal protection varied depending on chitosanases, suggesting that key amino acid residues contributed to resistance to heat denaturation. The transition temperatures (T(m)) of N174 were 54 degrees C and 69.5 degrees C in the absence and presence (6 g/l) of chitosan, respectively. T(m) were increased by 11.6 degrees C (N174-CONS), 13.8 degrees C (CSN-A104L), 15.6 degrees C (N174-K164R) and 25.2 degrees C (SCO) in the presence of chitosan (6 g/l). The thermal protective effect was attributed to an enzyme-ligand thermostabilization mechanism since it was not mimicked by the presence of anionic (carboxymethyl cellulose, heparin) or cationic (polyethylene imine) polymers, polyhydroxylated (glycerol, sorbitol) compounds or inorganic salts. Furthermore, the data from fluorometry experiments were in agreement with those obtained by analysis of reaction time-courses performed at 61 degrees C in which case CSN-A104L was rapidly inactivated whereas N174, N174-CONS and N174-K164R remained active over a reaction time of 90 min. This study presents evidence that (1) the fluorometric determination of T(m) in the presence of chitosan is a reliable technique for a rapid assessment of the thermal behavior of chitosanases, (2) it is applicable to structure-function studies of mutant chitosanases and, (3) it can be useful to provide an insight into the mechanism by which mutations can influence chitosanase stability.     

Reovirus μ2 protein modulates host cell alternative splicing by reducing protein levels of U5 snRNP core components

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family presenting a promising activity as an oncolytic virus. Recent studies have underlined MRV’s ability to alter cellular alternative splicing (AS) during infection, with a limited understanding of the mechanisms at play. In this study, we investigated how MRV modulates AS. Using a combination of cell biology and reverse genetics experiments, we demonstrated that the M1 gene segment, encoding the μ2 protein, is the primary determinant of MRV’s ability to alter AS, and that the amino acid at position 208 in μ2 is critical to induce these changes. Moreover, we showed that the expression of μ2 by itself is sufficient to trigger AS changes, and its ability to enter the nucleus is not required for all these changes. Moreover, we identified core components of the U5 snRNP (i.e. EFTUD2, PRPF8, and SNRNP200) as interactors of μ2 that are required for MRV modulation of AS. Finally, these U5 snRNP components are reduced at the protein level by both MRV infection and μ2 expression. Our findings identify the reduction of U5 snRNP components levels as a new mechanism by which viruses alter cellular AS.     

Group Choice as a Function of Group Size Differences and Assessment Time in Fish: The Influence of Species Vulnerability to Predation

The shoal-choice behaviour of two species of fish that differ in their vulnerability to predation was compared. Individuals of threespine stickleback, Gasterosteus aculeatus, and creek chub, Semotilus atromaculatus, were presented with a simultaneous choice of two equidistant stimulus shoals of conspecifics that differed in membership size (5 vs. 6 fish, 5 vs. 7, 5 vs. 8, 5 vs. 9 and 5 vs. 10). Test fish were allowed to view the stimulus shoals from a standard distance for either 10–20 or 120–150s before being frightened with a stimulus from an overhead light and released to join either shoal. We observed which shoal (the smaller or the larger one) the test fish approached. Preference for the larger stimulus shoal generally increased with increasing shoal size difference and with the duration of the assessment period, and was more pronounced in chub (the more vulnerable of the two species). For the short assessment period, chub showed a significantly stronger preference for the larger stimulus shoal than sticklebacks, whereas there was no significant difference between species for the long assessment period. Furthermore, chub responded more readily to small differences in shoal size (of 1–3 fish) than sticklebacks, for both short and long assessment periods. The above results are consistent with the hypothesis that chub, as the more vulnerable of the two species (in terms of predation), should be able to identify the larger of two shoals more quickly and should be more sensitive to small differences in shoal size than sticklebacks.