The methionine sulfoxide reductases: Catalysis and substrate specificities

Oxidation of Met residues in proteins leads to the formation of methionine sulfoxides (MetSO). Methionine sulfoxide reductases (Msr) are ubiquitous enzymes, which catalyze the reduction of the sulfoxide function of the oxidized methionine residues. In vivo, the role of Msrs is described as essential in protecting cells against oxidative damages and to play a role in infection of cells by pathogenic bacteria. There exist two structurally-unrelated classes of Msrs, called MsrA and MsrB, with opposite stereoselectivity towards the S and R isomers of the sulfoxide function, respectively. Both Msrs present a similar three-step catalytic mechanism. The first step, called the reductase step, leads to the formation of a sulfenic acid on the catalytic Cys with the concomitant release of Met. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the catalysis, in particular of the reductase step, and in structural specificities.     

Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.     

Cytotoxicity of the isoflavone genistein in NIH 3T3 cells

Genistein is one of the naturally occurring isoflavones present in plants such as soybeans and is commonly found in a variety of human foods. A number of studies indicated that this class of compounds exerts anticancerogenic and antimutagenic effects in various in vitro systems and in vivo animal models. We studied the effects of genistein on NIH 3T3 cells in in vitro models. The isoflavone genistein has been identified as having antiproliferative and apoptotic effects on various malignant cell types derived from solid tumors. Therefore, the cytotoxic and apoptotic properties of this compound were studied by MTT assay and Hoechst 33258/propidium iodide staining technique. The morphological changes of cells were examined in inverted fluorescent microscope. The oxidation of protein thiol groups and thiobarbituric-acid-reactive species (TBARS) was also determined. The cells were exposed to different concentrations of genistein (0-90 microM) after 24 h of incubation. The results revealed that genistein in concentrations higher than 20 microM significantly reduced cell viability, caused cell morphological changes and induced apoptotic and necrotic cell death. Oxidative modification of protein increased in the cells exposed to genistein in a dose- and time-dependent manner. In conclusion, our preliminary in vitro studies demonstrate the damaging effects of genistein on the mouse embryonic fibroblast cell line.     

1H, 13C, and 15N resonance assignments of the reduced and oxidized forms of Bacillus subtilis thiol peroxidase

Bacterial thiol peroxidases (Tpxs) are antioxidant enzymes which exist in various bacteria. Tpxs reduce the lipid hydroperoxides to protect the membrane lipid from destruction by reactive oxygen species. Tpxs are essential enzymes for bacterial anaerobic growth. Herein, we report the resonance assignments of ¹H, ¹³C, and ¹⁵N atoms in both the reduced and oxidized forms of Bacillus subtilis Tpx.     

3,5-Bis(3-dimethylaminomethyl-4-hydroxybenzylidene)-4-piperidone and related compounds induce glutathione oxidation and mitochondria-mediated cell death in HCT-116 colon cancer cells

This study aims at investigating the cytotoxicity and some of the modes of action of 3,5-bis(3-dimethylamino-4-hydroxybenzylidene)-4-piperidone trihydrochloride 3 and two related compounds 2 (which lacks the dimethylaminomethyl groups) and 4 (which has an additional dimethylaminoethyl substituent in both aryl rings) in order to ascertain the contribution of dimethylaminoethyl substituent to bioactivity. The bioactivities of 2–4 were compared with curcumin 5. Both 2 and 3 displayed submicromolar GI₅₀ values towards HCT-116 cells and were significantly more potent than 4, 5 and 5-fluorouracil (5-FU). All of the compounds displayed greater toxicity towards HCT-116 cells than human CRL-1790 non-malignant colon cells. In HCT-116 cells, the compounds 2, 3 and 5 increased the ratio of oxidised to reduced glutathione and destabilized the mitochondrial membrane potential. Both 2 and 5 produced an increase in mitochondrial superoxide and a burst in intracellular reactive oxygen species in HCT 116 cells. In addition, 2 and 4 stimulated respiration in rat liver mitochondria while 2 and 5 induced mitochondrial swelling. The results suggest that 2 and 5 cause oxidation or cross-linking of the thiols which control the mitochondrial permeability transition.     

Dynamic changes of red cell membrane thiol groups followed by bimane fluorescent labeling

Monobromobimane labels red cell membrane protein thiol groups; bands exhibit fluorescence after sodium dodecyl sulfate acrylamide gel electrophoresis and correspond to almost all of those staining with Coomassie blue. The response of membrane protein thiol groups to oxidative challenge and the dynamics of recovery of the thiol groups may be followed. Diminished labeling is found after oxidation with diamide, with both intrachain and interchain disulfide bond formation demonstrated by sodium dodecyl sulfate acrylamide gel electrophoresis. Regeneration of thiol groups under physiological conditions (incubation with glucose) after a moderate degree of diamide oxidation is shown to be complete (with respect to thiol group content and degree and distribution of bimane label) in normal human red blood cell membranes. Even after oxidation of almost half of the membrane protein thiol groups (maximum degree of oxidation achieved), regeneration of thiol groups is almost complete; a minor fraction resides in the form of disulfide-linked high molecular weight proteins (demonstrated by the electrophoretic profile) which may be reduced completely with dithiothreitol.Bimane fluorescent labeling provides a convenient and sensitive method for following membrane thiol group status under physiological conditions.     

Succinic semialdehyde dehydrogenase of wheat grain

Succinic semialdehyde dehydrogenase (EC 1.2.1.16) was purified 74-fold from wheat grain (Triticum durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both NAD and NADP support the oxidation of the substrate, but the former is 7-fold more active than the latter. The optimum pH for activity is around 9; the enzyme is stable in the pH range 6–9 and retains its whole activity up to 40°C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K _m for SSA and for NAD are 7.4x10^-6 M and 2x10^-4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000.Abbreviations GABA -aminobutyric acid - GABA-T -aminobutyric acid transaminase - ME mercaptoethanol - SSA succinic semialdehyde - SSA-DH succinic semialdehyde dehydrogenase     

On the role of S: Sulfotransferases in assimilatory sulfate reduction by plant cell suspension cultures

Cell suspension cultures of Catharanthus roseus (L.) G. Don were grown under S-auxotrophic (1.8 mM sulfate) and under S-heterotrophic (0.5 and 1.0 mM cysteine or methionine) conditions. The development of activity of the thiol sulfotransferases was followed during the complete growth period. Under auxotrophic growth, an NADPH-dependent S: sulfotransferase and a GSH-dependent S: sulfotransferase developed identically, whereas under heterotrophic growth, differences in the amount of enzymes and in the time course of their development occurred. The NADPH-dependent sulfotransferase appeared repressed by the S-amino acids but the GSH-dependent enzyme was derepressed. In that phenomenon, the development of the GSH sulfotransferase paralleled the development of the ATP-sulfurylase (EC 2774) activity of the cells.Abbreviations APS adenylylphosphosulfate - GSH reduced glutathione - PAPS phosphoadenylylphosphosulfate     

Redox sensing by Escherichia coli: effects of dithiothreitol, a redox reagent reducing disulphides, on bacterial growth

Escherichia coli is able to grow with a high rate under anaerobic conditions upon decrease in redox potential (E(h)) both either in slightly alkaline (pH 7.5) or acidic (pH 5.5) medium. Upon transition of E. coli MC4100 culture to stationary growth phase a decrease in E(h) from the positive values of +120 to +160 mV to the negative ones of -380 to -550 mV, and the H(2) production are observed at various pH. A redox reagent dl-dithiothreitol (DTT) in a concentration of 3mM reduces E(h) to the negative values, and increases a latent (lag) growth phase duration, as well as delays a logarithmic growth phase independently of pH. At alkaline and acidic pH the changes in membrane potential (DeltaPsi) are observed in the presence of 3mM DTT. K(+) uptake is recovered. At pH 5.5 the H(2) production is suppressed by DTT only in a higher concentration of 10 mM. The results suggest DTT effects that are in addition to the effects of E(h). The mechanism of DTT action on bacterial growth might be intermediated through thiol group modulation of the membrane proteins, which is reflected as the generation of DeltaPsi as well as K(+) accumulation and the activity of the membrane-associated enzymes.     

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.