SkQThy, a novel and promising mitochondria-targeted antioxidant.

Since thymoquinone (2-isopropyl-5-methylbenzoquinone) isolation from Nigella sativa in 1963, various studies have reported on its diverse pharmacological properties. However, despite its versatile healing abilities, clinical trials involving the use of thymoquinone have not been initiated due to its poor bioavailability. Many attempts have been made to improve the therapeutic efficacy of thymoquinone by synthesizing analogs, as well as by developing nanotechnology-based delivery systems. We hypothesized that some of the issues with thymoquinone delivery and bioavailability could be resolved by targeted delivery to mitochondria of thymoquinone derivatives conjugated to the penetrating lipophilic cationic triphenylphosphonium fragment. As mitochondria are the major site of reactive oxygen species generation in the cell, such a membranotropic thymoquinone derivative can act as an efficient antioxidant or prooxidant depending on the concentration used. Based on these theoretical considerations, a novel mitochondria-targeted compound, SkQThy, was synthesized and its effects on rat liver mitochondria and yeast cells were examined. SkQThy was found to exhibit pronounced antioxidant activity in mammalian mitochondria and yeast cells, decreasing hydrogen peroxide production in mitochondria, as well as preventing prooxidant-induced oxidative stress and mitochondrial fragmentation in yeast cells and increasing cell viability. Moreover, SkQThy proved itself to be the most efficient mitochondria-targeted antioxidant within the SkQs family, showing good therapeutic potential.

New Data on Effects of SkQ1 and SkQT1 on Rat Liver Mitochondria and Yeast Cells.

Mitochondria are involved in many processes in eukaryotic cells. They play a central role in energy conservation and participate in cell metabolism and signaling pathways. Mitochondria are the main source of reactive oxygen species, excessive generation of which provokes numerous pathologies and cell death. One of the most promising approaches to the attenuation of oxidative stress in mitochondria is the use of targeted (i.e., transported exclusively into mitochondria) lipophilic cationic antioxidants. These compounds offer advantages over conventional water-soluble antioxidants because they induce the so-called "mild uncoupling" and can prevent collapse of the membrane potential in low, nontoxic concentrations. A novel mitochondria-targeted antioxidant, SkQT1, was synthesized and tested within the framework of the research project guided by V. P. Skulachev. The results of these experiments were initially reported in 2013; however, one publication was not able to accommodate all the data on the SkQT1 interactions with isolated mitochondria and cells. Here, we examined comparative effects of SkQT1 and SkQ1 on rat liver mitochondria (with broader spectrum of energy parameters being studied) and yeast cells. SkQT1 was found to be less effective uncoupler, depolarizing agent, inhibitor of respiration and ATP synthesis, and "opener" of a nonspecific pore compared to SkQ1. At the same time SkQ1 exhibited higher antioxidant activity. Both SkQT1 and SkQ1 prevented oxidative stress and mitochondria fragmentation in yeast cells exposed to t-butyl hydroperoxide and promoted cell survival, with SkQT1 being more efficient than SkQ1. Together with the results presented in 2013, our data suggest that SkQT1 is the most promising mitochondria-targeted antioxidant that can be used for preventing various pathologies associated with the oxidative stress in mitochondria.

More about Interactions of Rhodamine 19 Butyl Ester with Rat Liver Mitochondria.

Oxidative stress is one of the major factors underlying mitochondrial dysfunctions. One of the most promising approaches for alleviating or preventing oxidative stress is the use of cationic uncouplers that accumulate in mitochondria in accordance to the level of the membrane potential, producing "mild" uncoupling. Based on this theoretical background, cationic rhodamine 19 butyl ester (C4R1) was synthesized and tested within the framework of the research project guided by V. P. Skulachev. The results of these tests were presented (Khailova et al. (2014) Biochim. Biophys. Acta, 1837, 1739-1747), but one publication could not accommodate all data on interactions of C4R1 with isolated mitochondria. In addition to previously presented data, we found that the effect of C4R1 on the rate of oxygen uptake is subject to temporal variations, which probably reflects variable rates of C4R1 entry into the mitochondria. Consequently, transient stimulation of respiration can be followed by inhibition. C4R1 was found not to shunt electron flow from complex I of the respiratory chain; it largely acted as an inhibitor of complex I in the respiratory chain and showed antioxidant activity. C4R1 taken at low, non-uncoupling concentrations enhanced the uncoupling activity of fatty acids (e.g. palmitate). Relatively low C4R1 concentrations stimulated opening of a nonspecific Ca2+/Pi-dependent pore. ATP synthesis and hydrolysis were substantially inhibited by C4R1 at low concentrations that had no appreciable effects on respiration in states 4 and 3 and only slightly decreased the membrane potential. Besides, conditions were revealed allowing correct evaluation of the membrane potential generated at the inner mitochondrial membrane with safranin O upon oxidation of both succinate and NAD-dependent substrates in the presence of C4R1.

Physiological role of alternative oxidase (from yeasts to plants).

Mitochondria of all so far studied organisms, with the exception of Archaea, mammals, some yeasts, and protists, contain, along with the classical phosphorylating cytochrome pathway, a so-called cyanide-insensitive alternative oxidase (AOX) localized on the matrix side of the mitochondrial inner membrane, and electron transport through which is not coupled with ATP synthesis and energy accumulation. Mechanisms underlying plentiful functions of AOX in organisms at various levels of organization ranging from yeasts to plants are considered. First and foremost, AOX provides a chance of cell survival after inhibiting the terminal components of the main respiratory chain or losing the ability to synthesize these components. The vitally important role of AOX is obvious in thermogenesis of thermogenic plant organs where it becomes the only terminal oxidase with a very high activity, and the energy of substrate oxidation by this respiratory pathway is converted into heat, thus promoting evaporation of volatile substances attracting pollinating insects. AOX plays a fundamentally significant role in alleviating or preventing oxidative stress, thus ensuring the defense against a wide range of stresses and adverse environmental conditions, such as changes in temperature and light intensities, osmotic stress, drought, and attack by incompatible strains of bacterial pathogens, phytopathogens, or their elicitors. Participation of AOX in pathogen survival during its existence inside the host, in antivirus defense, as well as in metabolic rearrangements in plants during embryogenesis and cell differentiation is described. Examples are given to demonstrate that AOX might be an important tool to overcome the adverse aftereffects of restricted activity of the main respiratory chain in cells and whole animals.

Alternative oxidase: distribution, induction, properties, structure, regulation, and functions.

The respiratory chain in the majority of organisms with aerobic type metabolism features the concomitant existence of the phosphorylating cytochrome pathway and the cyanide- and antimycin A-insensitive oxidative route comprising a so-called alternative oxidase (AOX) as a terminal oxidase. In this review, the history of AOX discovery is described. Considerable evidence is presented that AOX occurs widely in organisms at various levels of organization and is not confined to the plant kingdom. This enzyme has not been found only in Archaea, mammals, some yeasts and protists. Bioinformatics research revealed the sequences characteristic of AOX in representatives of various taxonomic groups. Based on multiple alignments of these sequences, a phylogenetic tree was constructed to infer their possible evolution. The ways of AOX activation, as well as regulatory interactions between AOX and the main respiratory chain are described. Data are summarized concerning the properties of AOX and the AOX-encoding genes whose expression is either constitutive or induced by various factors. Information is presented on the structure of AOX, its active center, and the ubiquinone-binding site. The principal functions of AOX are analyzed, including the cases of cell survival, optimization of respiratory metabolism, protection against excess of reactive oxygen species, and adaptation to variable nutrition sources and to biotic and abiotic stress factors. It is emphasized that different AOX functions complement each other in many instances and are not mutually exclusive. Examples are given to demonstrate that AOX is an important tool to overcome the adverse aftereffects of restricted activity of the main respiratory chain in cells and whole animals. This is the first comprehensive review on alternative oxidases of various organisms ranging from yeasts and protists to vascular plants.

Novel mitochondria-targeted compounds composed of natural constituents: conjugates of plant alkaloids berberine and palmatine with plastoquinone.

Novel mitochondria-targeted compounds composed entirely of natural constituents have been synthesized and tested in model lipid membranes, in isolated mitochondria, and in living human cells in culture. Berberine and palmatine, penetrating cations of plant origin, were conjugated by nonyloxycarbonylmethyl residue with the plant electron carrier and antioxidant plastoquinone. These conjugates (SkQBerb, SkQPalm) and their analogs lacking the plastoquinol moiety (C10Berb and C10Palm) penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in isolated mitochondria or in mitochondria in living human cells in culture. Reduced forms of SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations. In isolated mitochondria and in living cells, the berberine and palmatine moieties were not reduced, so antioxidant activity belonged exclusively to the plastoquinol moiety. In human fibroblasts, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide. At higher concentrations, conjugates of berberine and palmatine induced proton transport mediated by free fatty acids both in model and in mitochondrial membrane. In mitochondria this process was facilitated by the adenine nucleotide carrier. As an example of application of the novel mitochondria-targeted antioxidants SkQBerb and SkQPalm to studies of signal transduction, we discuss induction of cell cycle arrest, differentiation, and morphological normalization of some tumor cells. We suggest that production of oxygen radicals in mitochondria is necessary for growth factors-MAP-kinase signaling, which supports proliferation and transformed phenotype.

Interaction of tetraphenylphosphonium and dodecyltriphenylphosphonium with lipid membranes and mitochondria.

The permeability of a planar lipid membrane (composed of diphytanoylphosphatidylcholine) for tetraphenylphosphonium (TPP) was investigated. The observed level of the diffusion potential generated as a function of the TPP concentration gradient differed from the theoretically expected value, possibly due to proton leakage of the membrane mediated by the traces of fatty acids in the phospholipid forming the membrane. Using the molecular dynamics approach to study movement of TPP and dodecyltriphenylphosphonium (C(12)TPP) with different affinity to the lipid bilayer through a bilayer lipid membrane, it was found that C(12)TPP has a greater affinity to the membrane surface than TPP. However, the two cations have the same activation energy for transmembrane transfer. Interaction of TPP and C(12)TPP with tightly-coupled mitochondria from the yeast Yarrowia lipolytica was also investigated. At low, micromolar concentrations, both cations are "relatively weak, mild uncouplers", do not shunt electron transfer along the respiratory chain, do not disturb (damage) the inner mitochondrial membrane, and profoundly promote the uncoupling effect of fatty acids. At higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their detergent action.

Phenoptosis in yeasts.

The current view on phenoptosis and apoptosis as genetic programs aimed at eliminating potentially dangerous organisms and cells, respectively, is given. Special emphasis is placed on apoptosis (phenoptosis) in yeasts: intracellular defects and a plethora of external stimuli inducing apoptosis in yeasts; distinctive morphological and biochemical hallmarks accompanying apoptosis in yeasts; pro- and antiapoptotic factors involved in yeast apoptosis signaling; consecutive stages of apoptosis from external stimulus to the cell death; a prominent role of mitochondria and other organelles in yeast apoptosis; possible pathways for release of apoptotic factors from the intermembrane mitochondrial space into the cytosol are described. Using some concrete examples, the obvious physiological importance and expediency of altruistic death of yeast cells is shown. Poorly known aspects of yeast apoptosis and prospects for yeast apoptosis study are defined.