József Belágyi

Chromate sensitivity in fission yeast is caused by increased glutathione reductase activity and peroxide overproduction

The Cr(VI)‐sensitive mutant chr‐51S of the Schizosaccharomyces pombe accumulated chromate (CrO42–) and reduced Cr(V) to much greater extent, than did its parental strain 6 chr+. Sublethal doses of K2Cr2O7 did not induce any adaptive stress response, while H2O2 or menadione pretreatment proved protective against the cell injuries caused by Cr(VI). The intracellular GSH concentration in chr‐51S cells was approximately half of that for the 6 chr+. Moreover, the glutathione disulfide reducing capacity of chr‐51S was characterized by significantly increased glutathione reductase (GR) and glucose‐6‐phosphate dehydrogenase activities. These data strongly suggested that, instead of GSH, the NADPH/GR system was the major one‐electron Cr(VI) reductant in vivo. The increased Cr(V) reduction in chr‐51S mutant was accompanied by high intracellular superoxide and peroxide concentrations, required for formation of the hydroxyl radical (•OH). The decreased intracellular GSH levels and the Cr(VI)‐sensitive phenotype of the chr‐51S cells indicates that GSH might act effectively against chromate by scavenging •OH.

THE FLEXIBILITY OF ACTIN FILAMENTS AS REVEALED BY FLUORESCENCE RESONANCE ENERGY TRANSFER : THE INFLUENCE OF DIVALENT CATIONS

The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f′, was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility of the protein matrix within actin protomers (intramonomer flexibility). In both cases the relative increase as a function of temperature in f′ is larger in calcium-F-actin than in magnesium-F-actin in the range of 5–40 °C, which indicates that both the intramonomer and the intermonomer flexibility of the actin filaments are larger in calcium-F-actin than those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the magnesium-F-actin. The distance between Gln41 and Cys374 residues was found to be cation-independent and did not change during polymerization at 21 °C. The steady-state fluorescence anisotropy data of fluorophores attached to the Gln41 or Cys374 residues suggest that the microenvironments around these regions are more rigid in the magnesium-loaded actin filament than in the calcium-loaded form.

In vivo direct patulin-induced fluidization of the plasma membrane of fission yeast Schizosaccharomyces pombe.

Patulin is a toxic metabolite produced by various species of Penicillium, Aspergillus and Byssochlamys. In the present study, its effects on the plasma membrane of fission yeast Schizosaccharomyces pombe were investigated. The phase-transition temperature (G) of untreated cells, measured by electron paramagnetic resonance spectrometry proved to be 14.1 degrees C. Treatment of cells for 20 min with 50, 500, or 1000 microM patulin resulted in a decrease of the G value of the plasma membrane to 13.9, 10.1 or 8.7 degrees C, respectively. This change in the transition temperature was accompanied by the loss of compounds absorbing light at 260 nm. Treatment of cells with 50, 500 or 1000 microM patulin for 20 min induced the efflux of 25%, 30.5% or 34%, respectively, of these compounds. Besides its cytotoxic effects an adaptation process was observed. This is the first study to describe the direct interaction of patulin with the plasma membrane, a process which could definitely contribute to the adverse toxic effects induced by patulin.

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

Conformational and dynamic properties of actin filaments polymerized from ATP- or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys374 residue of actin was smaller in filaments from ADP-actin than in filaments from ATP-actin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys374 from the longitudinal filament axis. Temperature-dependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys374 of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.

The uncoupling of the effects of formins on the local and global dynamics of actin filaments

In this study, experiments were carried out in the conventional and saturation-transfer electron paramagnetic resonance (EPR) time domains to explore the effect of mDia1-FH2 formin fragments on the dynamic and conformational properties of actin filaments. Conventional EPR measurements showed that addition of formin to actin filaments produced local conformational changes in the vicinity of Cys-374 by increasing the flexibility of the protein matrix in the environment of the label. The results indicated that it was the binding of formin to the barbed end that resulted in these conformational changes. The conventional EPR results obtained with actin labeled on the Lys-61 site showed that the binding of formins could only slightly affect the structure of the subdomain 2 of actin, reflecting the heterogeneity of the formin-induced conformational changes. Saturation transfer EPR measurements revealed that the binding of formins decreased the torsional flexibility of the actin filaments in the microsecond time range. We concluded that changes in the local and the global conformational fluctuations of the actin filaments are associated with the binding of formins to actin. The results on the two EPR time domains showed that the effects of formins on the substantially different types of motions were uncoupled.

Regulation of patulin-induced oxidative stress processes in the fission yeast Schizosaccharomyces pombe.

Patulin (PAT), is one of the most widely disseminated mycotoxins found in agricultural products. In this study the PAT-induced accumulation of reactive oxygen species (ROS) and the regulation of the specific activities of antioxidant enzymes were investigated in the single cell eukaryotic organism Schizosaccharomyces pombe. In comparison with the untreated cells, 500 μM PAT treatment caused a 43% decrease in the concentration of the main intracellular antioxidant, glutathione (GSH); this depletion of GSH initiated a 2.44- and a 2.6-fold accumulation of superoxide anion and hydrogen peroxide, respectively, but did not increase the concentration of hydroxyl radicals; the reduction of ROS-induced adaptation processes via the activation of Pap1 transcription factor resulted in significantly increased specific activities of Cu/Zn superoxide dismutase, catalase and glutathione S-transferase to protect the cells against the ROS-induced unbalanced redox state. However, no change was measured in the activities of glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase. It seems reasonable to assume that the temporary PAT-induced ROS accumulation plays a crucial role in adaptation processes. The adverse effects of PAT may be exerted mainly through the destruction of cellular membranes and protein/enzyme functions.

Fluorescence quenching of the tryptophan emission from the F- and G-forms of actin

The parameters characterizing the quenching of fluorescence emitted by the four tryptophans of actin were measured by time resolved techniques in the monomeric (G) and the polymerized (F) forms of the protein. Acrylamide as a neutral and cesium ions as positively charged quenchers were used to characterize the subdomain 1, which contains all the four tryptophan residues. Use of acrylamide did not reveal any difference between the G- and F-forms, cesium, however, did so. The value of the quenching rate constant, k+, is significantly higher for the F-form than that for the G-form. This difference is present independently of the model (discrete or continuous lifetime distribution) used to process the data. These results are compatible with the conclusion that the charge distribution of the microenvironment around at least one of the four tryptophans is changed. This means that this region becomes more attracted to the cesium ion as a result of transition from the G- to the F-form.

DSC and EPR study on AMP.PNP, BeFx and AlF4 containing myosin nucleotide complexes

Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated skeletal muscle fibres to study the effect of the binding of nucleotides and nucleotide analogues to myosin. The thermal unfolding of muscle fibres in rigor showed three discrete domain regions with thermal stability of 52.2, 58.8 and 67.8°C. AMP.PNP and ATP plus AlF3 or BeF2 affected markedly the transitions, which implies the strong interaction between AMP.PNP or nucleotide analogues and catalytic domain of myosin, and a partial dissociation of heads from actin. ADP.BeFx and states model the transition states of the ATP hydrolysis cycle which precede the powerstroke of the muscle fibres. Spectrum deconvolution on isothiocyanate-labelled fibres in AMP.PNP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The myosin heads which showed high degree of order were in the strongly binding ADP-state. The spectra in - and ADP.BeFx state reflected random orientation of labels with increased rotational mobility in comparison with rigor. The results suggest that myosin in muscle fibres in ADP.BeFx state exists in two forms.

Adaptation to tert-butyl hydroperoxide at a plasma membrane level in the fission yeast Schizosaccharomyces pombe parental strain and its t-BuOOH-resistant mutant.

The one‐gene mutant hyd1‐190 of the fission yeast Schizosaccharomyces pombe displayed four‐fold resistance to tert‐butyl hydroperoxide (t‐BuOOH) in comparison with its parental strain hyd+. The cells of hyd1‐190 exhibited a quantitative alteration in the sterol content and hence in the fatty acid composition of the plasma membrane, reflected in a two‐fold amphotericin B sensitivity, increased rigidity of the plasma membrane, revealed by an elevated (Δ7.9 °C) phase‐transition temperature, measured by means of electron paramagnetic resonance spectroscopy, and a significantly decreased uptake of glycerol. Treatment of the strains with a subinhibitory concentration (0.2 mM) of t‐BuOOH induced adaptation via modification of the sterol and fatty acid compositions, resulting in increased (Δ3.95 °C) and decreased (Δ6.83 °C) phase‐transition temperatures of the hyd+ and hyd1‐190 strains, respectively, in order to defend the cells against the consequences of t‐BuOOH‐induced external oxidative stress. However, in contrast with hyd+, hyd1‐190 lacks the ability to adapt to t‐BuOOH at a cell level.

Oxidative stress induced by HIV-1 F34IVpr in Schizosaccharomyces pombe is one of its multiple functions

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during infection, including induction of cell cycle G(2) arrest and cell death in both human and the fission yeast Schizosaccharomyces pombe cells. In this study, a mutant derivative of Vpr (F34IVpr), which causes transient G2 arrest with little or no effect of cell killing, was used to study the molecular impact of Vpr on cellular oxidative stress responses in S. pombe. We demonstrated here that F34IVpr triggers low level of complex and atypical oxidative stress responses in comparison with its parental strain SP223 in early (14-h) and late (35-h) log phase cultures. Specifically, F34IVpr production in S. pombe causes significantly elevated levels of reactive oxygen species such as superoxide and peroxides; meanwhile, it also induces decreased levels of glutathione, hydroxyl radical concentrations and specific enzyme activities such as those of antioxidant enzymes including superoxide dismutases, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione transferase. These observations may provide functional insights into the significance of Vpr-induced oxidative stress as part of the multifaceted functions of Vpr, and contribute to the development of future new strategies aimed to reduce the adverse Vpr-mediated effects in HIV-infected patients.