R. A. Zvyagilskaya

Cross-talk between reactive oxygen species and calcium in living cells.

The results of many investigations have shown that calcium is essential for production of reactive oxygen species (ROS). Elevation of intracellular calcium level is responsible for activation of ROS-generating enzymes and formation of free radicals by the mitochondria respiratory chain. On the other hand, an increase in intracellular calcium concentration may be stimulated by ROS. H2O2 has been recently shown to accelerate the overall channel opening process in voltage-dependent calcium channels in plant and animal cells. The 1,4,5-inositol-triphosphate-receptors as well as the ryanodine receptors of sarcoplasmic reticulum have also been demonstrated to be redox-regulated. Activity of Ca2+-ATPases and Na2+/Ca2+ exchangers of animal cells are modulated by the intracellular redox state. Simultaneously, Ca2+ may activate antioxidant enzymes, such as plant catalase and glutathione reductase, and increase the level of superoxide dismutase in animal cells. Reviewed data support the speculation that Ca2+ and ROS are two cross-talking messengers in various cellular processes.

Prevention of cardiolipin oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs)

The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273-1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663-668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH2 interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ- being reduced back to SkQH2 by heme bH of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C9 or C13 of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (capital ES, Cyrillic12TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since capital ES, Cyrillic12TPP is specifically accumulated in mitochondria and increases the H+ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by capital ES, Cyrillic12TPP.

Apoptosis in Unicellular Organisms: Mechanisms and Evolution

Data about the programmed death (apoptosis) in unicellular organisms, from bacteria to ciliates, are discussed. Firstly apoptosis appeared in lower eukaryotes, but its mechanisms in these organisms are different from the classical apoptosis. During evolution, the apoptotic process has been improving gradually, with reactive oxygen species and Ca2+ playing an essential role in triggering apoptosis. All eukaryotic organisms have apoptosis inhibitors, which might be introduced by viruses. In the course of evolution, caspases and apoptosis-inducing factor appeared before other apoptotic proteins, with socalled death receptors being the last among them. The functional analogs of eukaryotic apoptotic proteins take parts in the programmed death of bacteria.

High-resolution structural analysis of a novel octaheme cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens

Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.

Nitrate reductases: structure, functions, and effect of stress factors.

Structural and functional peculiarities of four types of nitrate reductases are considered: assimilatory nitrate reductase of eukaryotes, as well as cytoplasmic assimilatory, membrane-bound respiratory, and periplasmic dissimilatory bacterial nitrate reductases. Arguments are presented showing that eukaryotic organisms are capable of nitrate dissimilation. Data concerning new classes of extremophil nitrate reductases, whose active center does not contain molybdocofactor, are summarized.

In search of novel highly active mitochondria-targeted antioxidants: thymoquinone and its cationic derivatives.

Since the times of the Bible, an extract of black cumin seeds was used as a medicine to treat many human pathologies. Thymoquinone (2‐demethylplastoquinone derivative) was identified as an active antioxidant component of this extract. Recently, it was shown that conjugates of plastoquinone and penetrating cations are potent mitochondria‐targeted antioxidants effective in treating a large number of age‐related pathologies. This review summarizes new data on the antioxidant and some other properties of membrane‐penetrating cationic compounds where 2‐demethylplastoquinone substitutes for plastoquinone. It was found that such a substitution significantly increases a window between anti‐ and prooxidant concentrations of the conjugates. Like the original plastoquinone derivatives, the novel compounds are easily reduced by the respiratory chain, penetrate through model and natural membranes, specifically accumulate in mitochondria in an electrophoretic fashion, and strongly inhibit H2O2‐induced apoptosis at pico‐ and nanomolar concentrations in cell cultures. At present, cationic demethylplastoquinone derivatives appear to be the most promising mitochondria‐targeted drugs of the quinone series.

Characterization of the Pho89 phosphate transporter by functional hyperexpression in Saccharomyces cerevisiae

The Na(+)-coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and special properties. In this study, we have used a pho84Deltapho87Deltapho90Deltapho91Delta quadruple deletion strain of S. cerevisiae devoid of all transporter genes specific for inorganic phosphate, except for PHO89, to functionally characterize Pho89 under conditions where its expression is hyperstimulated. Under these conditions, the Pho89 protein is strongly upregulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0, the transporter is functionally active under alkaline conditions only, with a K(m) value reflecting high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even under these hyperexpressive conditions, Pho89 is unable to sense and signal extracellular phosphate levels. In cells grown at pH 8.0, Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na(+) ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H(+)- and Na(+)-coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na(+)-coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH.

Proton- and Sodium-Coupled Phosphate transport Systems and Energy Status of Yarrowia lipolytica Cells Grown in Acidic and Alkaline Conditions

Abstract. In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5–10.5), which makes it an excellent model system for studying H+- and Na+-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5–10.0. In cells grown at pH 4.5, inorganic phosphate (Pi) was accumulated by two kinetically discrete H+/Pi-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high Pi concentrations. The high-affinity system, subjected to regulation by both extracellular Pi availability and intracellular polyphosphate stores, is mobilized during Pi-starvation. In cells grown at pH 9.5–10, Pi uptake is mediated by several kinetically discrete Na+-dependent systems that are specifically activated by Na+ ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high Pi concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during Pi-starvation and appear to be controlled by extracellular Pi. They represent the first examples of high-capacity, Na+-driven Pi transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H+- and Na+-coupled Pi transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H+-coupled Pi transport systems are predominant. The contribution of the Na+/Pi cotransport systems to the total cellular Pi uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher.

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca2+ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca2+ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca2+ overload. However, exposure of yeast mitochondria to 50–100 µM Ca2+ in the presence of the Ca2+ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca2+/nH+-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca2+- ETH129-induced activation of the Ca2+/H+-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca2+ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca2+ uptake and is differently regulated compared to the mPTP of animal mitochondria.

Isolation and Characterization of a Novel Leaf-Inhabiting Osmo-, Salt-, and Alkali-Tolerant Yarrowia lipolytica Yeast Strain

Salt‐excreting leaves of Atriplex halimus plants harvested in the central Negev Highlands of Israel were screened for yeasts inhabiting their surfaces. Several aerobic, moderately salt‐ and alkalitolerant yeasts were isolated. One of the isolates (tentatively designated S‐8) was identified as Yarrowia lipolytica (Wick.) van der Walt and Arx, on the basis of its morphological, biochemical/physiological characteristics, and of quantitative chemotaxonomic and molecular marker analyses. However, the strain is distinguished from the known members of the type Y. lipolytica strain by its pronounced osmo‐, salt‐, and pH tolerance. Cells displayed a unique capacity to grow over a wide pH range (from 3.5 to 11.5) with a pH optimum at 4.5 to 7.5. It is proposed that the S‐8 strain be assigned to a single Y. lipolytica species as its anamorpha, or as a new variety, Y. lipolytica var. alkalitolerance. The ecophysiological properties and biotechnological potentials of the new strain are discussed.