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Significance The mitochondria, organelles that produce the largest amounts of ATP and reactive oxygen species (mROS) in living cells, are equipped with a universal mechanism that can completely prevent mROS production. This mechanism consists of mild depolarization of the inner mitochondrial membrane to decrease the membrane potential to a level sufficient to form ATP but insufficient to generate mROS. In short-lived mice, aging is accompanied by inactivation of the mild depolarization mechanism, resulting in chronic poisoning of the organism with mROS. However, mild depolarization still functions for many years in long-lived naked mole rats and bats., The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H+-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.

Naked mole-rats express many unusual traits for such a small rodent. Their morphology, social behaviour, physiology, and ageing have been well studied over the past half-century. Many early findings and speculations about this subterranean species persist in the literature, although some have been repeatedly questioned or refuted. While the popularity of this species as a natural-history curiosity, and oversimplified story-telling in science journalism, might have fuelled the perpetuation of such misconceptions, an accurate understanding of their biology is especially important for this new biomedical model organism. We review 28 of these persistent myths about naked mole-rat sensory abilities, ecophysiology, social behaviour, development and ageing, and where possible we explain how these misunderstandings came about.

It has been suggested that highly social mammals, such as naked mole rats and humans, are long-lived due to neoteny (the prolongation of youth). In both species, aging cannot operate as a mechanism facilitating natural selection because the pressure of this selection is strongly reduced due to 1) a specific social structure where only the “queen” and her “husband(s)” are involved in reproduction (naked mole rats) or 2) substituting fast technological progress for slow biological evolution (humans). Lists of numerous traits of youth that do not disappear with age in naked mole rats and humans are presented and discussed. A high resistance of naked mole rats to cancer, diabetes, cardiovascular and brain diseases, and many infections explains why their mortality rate is very low and almost age-independent and why their lifespan is more than 30 years, versus 3 years in mice. In young humans, curves of mortality versus age start at extremely low values. However, in the elderly, human mortality strongly increases. High mortality rates in other primates are observed at much younger ages than in humans. The inhibition of the aging process in humans by specific drugs seems to be a promising approach to prolong our healthspan. This might be a way to retard aging, which is already partially accomplished via the natural physiological phenomenon neoteny.

Age-related dysfunctions are accompanied by impairments in the mitochondrial morphology, activity of signaling pathway, and protein interactions. Cardiolipin is one of the most important phospholipids that maintains the curvature of the cristae and facilitates assembly and interaction of complexes and supercomplexes of the mitochondrial respiratory chain. The fatty acid composition of cardiolipin influences the biophysical properties of the membrane and, therefore, is crucial for the mitochondrial bioenergetics. The presence of unsaturated fatty acids in cardiolipin is the reason of its susceptibility to oxidative damage. Damaged cardiolipin undergoes remodeling by phospholipases, acyltransferases, and transacylases, creating a highly specific fatty acyl profile for each tissue. In this review, we discuss the variability of cardiolipin fatty acid composition in various species and different tissues of the same species, both in the norm and at various pathologies (e.g., age-related diseases, oxidative and traumatic stresses, knockouts/knockdowns of enzymes of the cardiolipin synthesis pathway). Progressive pathologies, including age-related ones, are accompanied by cardiolipin depletion and decrease in the efficiency of its remodeling, as well as the activation of an alternative way of pathological remodeling, which causes replacement of cardiolipin fatty acids with polyunsaturated ones (e.g., arachidonic or docosahexaenoic acids). Drugs or special diet can contribute to the partial restoration of the cardiolipin acyl profile to the one rich in fatty acids characteristic of an intact organ or tissue, thereby correcting the consequences of pathological or insufficient cardiolipin remodeling. In this regard, an urgent task of biomedicine is to study the mechanism of action of mitochondria-targeted antioxidants effective in the treatment of age-related pathologies and capable of accumulating not only in vitro, but also in vivo in the cardiolipin-enriched membrane fragments.

Inverted repeats are common DNA elements, but they rarely overlap with protein-coding sequences due to the ensuing conflict with the structure and function of the encoded protein. We discovered numerous perfect inverted repeats of considerable length (up to 284 bp) embedded within the protein-coding genes in mitochondrial genomes of four Nematomorpha species. Strikingly, both arms of the inverted repeats encode conserved regions of the amino acid sequence. We confirmed enzymatic activity of the respiratory complex I encoded by inverted repeat-containing genes. The nucleotide composition of inverted repeats suggests strong selection at the amino acid level in these regions. We conclude that the inverted repeat-containing genes are transcribed and translated into functional proteins. The survey of available mitochondrial genomes reveals that several other organisms possess similar albeit shorter embedded repeats. Mitochondrial genomes of Nematomorpha demonstrate an extraordinary evolutionary compromise where protein function and stringent secondary structure elements within the coding regions are preserved simultaneously.

Oxidative stress and mitochondrial dysfunction are the key links in the chain of development of pathologies associated with the violation of cellular energy metabolism. Development of mitochondria-addressed compounds highly specific for chemical processes is one of the most promising ways to develop approaches to the treatment of inherited and age-related diseases with mitochondrial etiology. Correlation of structure and chemical activity of the test compounds from a class of lipophilic cations revealed the key role of substituents in the aromatic ring of 1,4-benzoquinones in the manifestation of high antioxidant properties. In this work, it is shown that a synthesized benzoquinone derivative conjugated in position 6 with membrane-penetrating cation of decyltriphenylphosphonium and with substituents at position 2, 3, and 5 (SkBQ) has much lower antioxidant and significantly higher prooxidant activity in comparison with similar derivatives of plasto- and toluquinone SkQ1 and SkQT1 in experiments on isolated mitochondria. At the same time, SkBQ, like SkQ1 and SkQT1, can be reduced by the respiratory chain in the center i of complex III and decrease the mitochondrial membrane potential. In cell cultures of human fibroblasts, it was revealed that SkBQ does not protect cells from apoptosis induced by hydrogen peroxide. Under the same conditions, SkQ1 and SkQT1 exhibit a powerful protective effect. Thus, SkBQ can be seen as a mitochondria-addressed prooxidant. The possibility of using SkBQ as an anticancer drug for the treatment of cancers such as prostate cancer whose cells are sensitive to mitochondrial reactive oxygen species is discussed.

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.

MtDNA mutator mice exhibit marked features of premature aging. We find that these mice treated from age of ≈100 days with the mitochondria-targeted antioxidant SkQ1 showed a delayed appearance of traits of aging such as kyphosis, alopecia, lowering of body temperature, body weight loss, as well as ameliorated heart, kidney and liver pathologies. These effects of SkQ1 are suggested to be related to an alleviation of the effects of an enhanced reactive oxygen species (ROS) level in mtDNA mutator mice: the increased mitochondrial ROS released due to mitochondrial mutations probably interact with polyunsaturated fatty acids in cardiolipin, releasing malondialdehyde and 4-hydroxynonenal that form protein adducts and thus diminishes mitochondrial functions. SkQ1 counteracts this as it scavenges mitochondrial ROS. As the results, the normal mitochondrial ultrastructure is preserved in liver and heart; the phosphorylation capacity of skeletal muscle mitochondria as well as the thermogenic capacity of brown adipose tissue is also improved. The SkQ1-treated mice live significantly longer (335 versus 290 days). These data may be relevant in relation to treatment of mitochondrial diseases particularly and the process of aging in general.

Plastoquinone, a very effective electron carrier and antioxidant of chloroplasts, was conjugated with decyltriphenylphosphonium to obtain a cation easily penetrating through membranes. This cation, called SkQ1, is specifically targeted to mitochondria by electrophoresis in the electric field formed by the mitochondrial respiratory chain. The respiratory chain also regenerates reduced SkQ1H(2) from its oxidized form that appears as a result of the antioxidant activity of SkQ1H(2). SkQ1H(2) prevents oxidation of cardiolipin, a mitochondrial phospholipid that is especially sensitive to attack by reactive oxygen species (ROS). In cell cultures, SkQ1 and its analog plastoquinonyl decylrhodamine 19 (SkQR1) arrest H(2)O(2)-induced apoptosis. When tested in vivo, SkQs (i) prolong the lifespan of fungi, crustaceans, insects, fish, and mice, (ii) suppress appearance of a large number of traits typical for age-related senescence (cataract, retinopathies, achromotrichia, osteoporosis, lordokyphosis, decline of the immune system, myeloid shift of blood cells, activation of apoptosis, induction of β-galactosidase, phosphorylation of H2AX histones, etc.) and (iii) lower tissue damage and save the lives of young animals after treatments resulting in kidney ischemia, rhabdomyolysis, heart attack, arrhythmia, and stroke. We suggest that the SkQs reduce mitochondrial ROS and, as a consequence, inhibit mitochondria-mediated apoptosis, an obligatory step of execution of programs responsible for both senescence and fast "biochemical suicide" of an organism after a severe metabolic crisis.

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.

A unique phenomenon of mitochondria-targeted protonophores is described. It consists in a transmembrane H + -conducting fatty acid cycling mediated by penetrating cations such as 10-(6’-plastoquinonyl)decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C 12 TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (ΔpH) to a membrane potential (Δ ψ ) of the Nernstian value (about 60 mV Δ ψ at ΔpH = 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C 12 TPP, but not cetyltrimethylammonium, potentiated fatty acid-induced ( i ) uncoupling of respiration and phosphorylation, and ( ii ) inhibition of H 2 O 2 formation. In intact yeast cells, C 12 TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanide phenylhydrazone) stimulated respiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization.

Mouse embryonic fibroblasts (MEF) with point mutation in somatic cytochrome C gene were generated and characterized. It was shown that the substitution of lysine for tryptophan in position 72 (K72W) decreased the proapoptotic functions of cytochrome C in response to staurosporin treatment without disrupting its respiratory functions. The presence of this mutation did not affect the pattern of cytochrome C gene expression or its localization inside the cell. These cell lines therefore represent an interesting model for the study of apoptotic signaling and physiological functions of cytochrome C.

For many pathological conditions, reactive oxygen species (ROS) generated in mitochondria are considered to have a role as a trigger. When mitochondrial ROS (mROS) are formed in the inner mitochondrial membrane, they initiate free radical-mediated chain reactions of lipid peroxidation and are thus especially damaging. The consequences of membrane damage are decreased electrical resistance of the membrane, oxidative damage to cardiolipin (a mitochondria specific lipid essential for functioning of respiratory chain proteins and H(+)-ATP synthase), and damage to mitochondrial DNA localized in close vicinity to the inner membrane, with consequent mitochondrial dysfunction and induction of apoptotic cascade and cell death. To target the starting point of such undesirable events, antioxidants conjugated with mitochondria-targeted, membrane-penetrating cations can be used to scavenge ROS inside mitochondria. The most demonstrative indications favoring this conclusion originate from recent discoveries of the in vivo effects of such cations belonging to the MitoQ and SkQ groups. Here we describe some essential methodological aspects of the application of mitochondria-targeted cations promising in treating oxidative stress-related pathologies.

Chemical and physiological functions of molecular oxygen and reactive oxygen species (ROS) and existing equilibrium between pools of pro-oxidants and anti-oxidants providing steady state ROS level vital for normal mitochondrial and cell functioning are reviewed. The presence of intracellular oxygen and ROS sensors is postulated and few candidates for this role are suggested. Possible involvement of ROS in the process of fragmentation of mitochondrial reticulum made of long mitochondrial filaments serving in the cell as "electric cables", as well as the role of ROS in apoptosis and programmed mitochondrial destruction (mitoptosis) are reviewed. The critical role of ROS in destructive processes under ischemia/reoxygenation and ischemic preconditioning is discussed. Mitochondrial permeability transition gets special consideration as a possible component of the apoptotic cascade, resulting in excessive "ROS-induced ROS release".

VDAC changes its structure either voltage dependent in artificial membranes or physiologically by interaction with the c conformation of the adenine nucleotide translocator (ANT). This interaction creates contact sites and leads to a specific organisation of cytochrome c in the VDAC ANT complexes. The VDAC structure specific for contact sites thus generates a signal at the surface for several proteins in the cytosol to bind with high affinity such as hexokinase, glycerolkinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC ANT complex has two critical qualities: firstly, external Bax gets access to the cytochrome c and secondly the ANT stays in the c conformation that easily changes the structure to an unspecific uni-porter causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as specific anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly hinders direct interaction between VDAC and ANT and secondly changes porin structure into low affinity for hexokinase and external Bax. Cytochrome c in the creatine kinase complex will be differently organised not accessible to Bax and the ANT is run as anti-porter by the active octamer. However, when free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax dependent cytochrome c release and risk of permeability transition pore opening.

The outer mitochondrial membrane pore (VDAC) changes its structure either voltage-dependently in artificial membranes or physiologically by interaction with the adenine nucleotide translocase (ANT) in the c-conformation. This interaction creates contact sites and leads in addition to a specific organisation of cytochrome c in the VDAC-ANT complexes. The VDAC structure that is specific for contact sites generates a signal at the surface for several proteins in the cytosol to bind with high capacity, such as hexokinase, glycerol kinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC-ANT complex has two critical qualities: firstly, Bax gets access to cytochrome c and secondly the ANT is set in its c-conformation that easily changes conformation into an unspecific channel (uniporter) causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly restrains interaction between VDAC and ANT and secondly changes the VDAC structure into low affinity for hexokinase and Bax. Cytochrome c in the creatine kinase complex will be differently organised, not accessible to Bax and the ANT is run as anti-porter by the active creatine kinase octamer. However, when, for example, free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax-dependent cytochrome c release and risk of permeability transition pore opening.

The release of cytochrome c from the intermembrane space of mitochondria into the cytosol is one of the critical events in apoptotic cell death. In the present study, it is shown that release of cytochrome c and apoptosis induced by tumor necrosis factor alpha (TNF) in HeLa cells can be inhibited by (i) overexpression of an oncoprotein Bcl-2, (ii) Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (PTP) or (iii) oligomycin, an inhibitor of H+- ATP-synthase. Staurosporine-induced apoptosis is sensitive to Bcl-2 but insensitive to Cyclosporin A and oligomycin. The effect of oligomycin is not due to changes in mitochondrial membrane potential or to inhibition of ATP synthesis/hydrolysis since (a) uncouplers (CCCP, DNP) which discharge the membrane potential fail to abolish the protective action of oligomycin and (b) aurovertin B (another inhibitor of H+-ATP-synthase, affecting its F1 component) do not affect apoptosis. A role of oligomycin-sensitive F0 component of H+-ATP-synthase in the TNF-induced PTP opening and apoptosis is suggested.

The mechanism by which external Bax releases cytochrome c is still controversial and may also depend on the type of mitochondria and the actual localisation of cytochrome c. Outer membrane porin acquires high binding affinity for hexokinase by interacting with the adenine nucleotide translocator (ANT) in the contact sites. (I) The hexokinase protein was thus used as a tool to isolate the contact site forming complex between outer membrane porin and inner membrane ANT from a TritonX100 extract of brain membranes. (II) A significant amount of cytochrome c was co-purified with the isolated hexokinase porin ANT complexes that were reconstituted in phospholipid vesicles. Bax-δC released the endogenous cytochrome c from the vesicles without forming unspecific pores. This was shown by loading the vesicles with malate that was not liberated by Bax-δC. (III) The Bax-δC effect was dependent on a specific association of cytochrome c with the porin ANT complex, as dissociation of the complex by bongkrekate abolished the Bax dependent cytochrome c liberation. (IV) The Bax-δC effect was as well suppressed by hexokinase phosphorylating glucose.

The mechanism by which the proapoptotic protein Bax releases cytochrome c from mitochondria is not fully understood. The present work approaches this problem using C-terminal truncated oligomeric Bax (BaxDeltaC). Micromolar concentrations of BaxDeltaC released cytochrome c from isolated rat heart and liver mitochondria, while the release of adenylate kinase was not significantly affected. BaxDeltaC also released cytochrome c but not adenylate kinase from outer membrane vesicles filled with these proteins. However, BaxDeltaC was ineffective in releasing cytochrome c when outer membrane vesicles were obtained in the presence of glycerol, conditions under which the number of contact sites was drastically reduced. BaxDeltaC did not liberate encapsulated cytochrome c and adenylate kinase from pure phospholipid vesicles or vesicles reconstituted with porin. However, when the hexokinase-porin-adenine nucleotide translocase complex from brain mitochondria was reconstituted in vesicles, BaxDeltaC released internal cytochrome c but not adenylate kinase. In all these systems, only a small portion of total cytochrome c present in either mitochondria or vesicles could be liberated by BaxDeltaC. BaxDeltaC also increased the accessibility of external cytochrome c to either oxidation by complex IV or reduction by complex III in intact liver and heart mitochondria.(1) BaxDeltaC selectively releases cytochrome c and enables a bidirectional movement of cytochrome c across the outer mitochondrial membrane. (2) A multiprotein complex that resembles the mitochondrial contact sites is a prerequisite for BaxDeltaC action. (3) A limited pool of cytochrome c becomes the first target for BaxDeltaC.

Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin–ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intra-mitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein.

Bacterial luciferases are highly suitable test substrates for the analysis of refolding of misfolded proteins, as they are structurally labile and loose activity at 42 degrees C. Heat-denatured thermolabile Vibrio fischeri luciferase and thermostable Photorhabdus luminescens luciferase were used as substrates. We found that their reactivation requires the activity of the DnaK chaperone system. The DnaKJ chaperones were dispensable in vivo for de novo folding at 30 degrees C of the luciferase, but essential for refolding after a heat-shock. The rate and yield of DnaKJ refolding of the P. luminescens thermostable luciferase were to a marked degree lower as compared with the V. fischeri thermolabile luciferase. The refolding activity of the DnaKJ chaperones was examined at various temperatures. Between 30 and 37 degrees C, the refolding rates of the V. fischeri luciferase decreased and the reaction reached a complete arrest at temperatures above 40 degrees C. The rate of DnaKJ-mediated refolding of the thermostable luciferase at first increased between 30 and 37 degrees C and then decreased at the range of 37-44 degrees C. We observed that the rate of DnaKJ-mediated refolding of the heat-denatured P. luminescens thermostable luciferase, but not of the thermolabile V. fischeri luciferase, decreased during the prolonged incubation at a high (47 degrees C) temperature. The efficiency and reversibility of protein refolding arrest during and after heat-shock strongly depended on the stability of the DnaKJ-denatured luciferase complex. It is supposed that the thermostable luciferase is released during the heat-shock, whereas the thermolabile luciferase remained bound to the chaperone.

The role of oxygen in the induction of mitochondrial permeability transitions was studied. Oxygen consumption, swelling, membrane potential and calcium transport were recorded simultaneously in isolated rat liver mitochondria. Oxygen depletion was accomplished by saturating the medium with N2 and allowing either mitochondrial respiration or glucose/glucose oxidase to consume the residual oxygen. Upon anaerobiosis, mitochondria were supplemented with 500 microM ATP to support succinate-driven membrane potential. Under these conditions, 100 microM Ca2+ induced cyclosporin A-sensitive permeability transitions. To eliminate the possible inhibition of permeability transition by high concentrations of adenine nucleotides, anaerobic mitochondria were also energized by the combination of 20 microM ADP and phosphoenolpyruvate/pyruvate kinase. These mitochondria also underwent Ca2+-induced permeability transition. Under both of these conditions, namely the addition of ATP as a single or through actions of pyruvate kinase, the respiratory components were totally reduced. Thus, oxygen is not a necessary factor for mitochondria to undergo permeability transitions.

Originally published in: Bacterial and Eukaryotic Porins. Edited by Roland Benz. Copyright © 2005 Wiley-VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-30775-3 The sections in this article are Introduction Structure and Isotypes of VDAC The Influence of Phospholipids on VDAC Structure VDAC Conductance and Ion Selectivity Physiological Significance of the Voltage Dependence Porins as Specific Binding Sites VDAC senses Inner Membrane Functions in the Contact Site Cytochrome c is a Component of the Contact Sites Isolation and Characterization of VDAC-ANT Complexes Reconstitution of VDAC-ANT Complexes Importance of Metabolic Channeling in Regulation of Energy Metabolism The VDAC-ANT Complex as Permeability Transition Pore The VDAC-ANT Complex as a Target for Bax-dependent Cytochrome c Release The VDAC–ANT Complexes contain Cytochrome c The Importance of the Kinases in Regulation of Apoptosis Suppression of Bax-dependent Cytochrome c Release and Permeability Transition by Hexokinase Suppression of Permeability Transition by Mitochondrial Creatine Kinase Conclusion Keywords: function of the outer mitochondrial membrane pore in intracellular signaling; structure and isotypes of VDAC; influence of phospholipids on VDAC structure; VDAC conductance and ion selectivity; porins as specific binding sites; cytochromec; VDAC-ANT complexes

In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199-208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (Delta Psi), the compartment with a lower dication concentration positive. However, the Delta Psi values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at Delta Psi >100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian Delta Psi, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered Delta Psi. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria.

Reoxygenation following ischemia causes tissue oxidative stress. We studied the role of oxidative stress caused by kidney ischemia/reperfusion (I/R) on the mitochondria of renal tissue slices. I/R caused the mitochondria to be swollen, fragmented, and have lower membrane potential. The mitochondria generated more reactive oxygen species (ROS) and nitric oxide (NO) in situ as measu red by fluorescence of ROS- and NO-sensitive probes. Infusion of lithium ion, an inhibitor of glycogen kinase synthase-3, caused phosphorylation of its Ser-9 and restored the membrane potential and decreased ROS production of the mitochondrial fraction. Ischemic kidney and hypoxic rat preconditioning improved mitochondrial membrane potential and lowered ROS production caused by subsequent I/R similar to lithium ion infusion. Preconditioning normalized NO production in mitochondria as well. The drop in the mitochondrial membrane potential was prevented by NO synthase inhibition, demonstrating a strong contribution of NO to changes in mitochondrial energy metabolism during the I/R transition. Mitochondria in the I/R-stressed kidney contained less cytochrome c and more pro-apoptotic Bax, consistent with apoptotic degradation.

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1 = SkQR1 > SkQ3 > MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H2O2-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H2O2 or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53−/− mice, 5 nmol/kg × day SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.

The effect of the mitochondria-targeted, plastoquinone-containing antioxidant SkQ1 on the lifespan of outbred mice and of three strains of inbred mice was studied. To this end, low pathogen (LP) or specific pathogen free (SPF) vivaria in St. Petersburg, Moscow, and Stockholm were used. For comparison, we also studied mole-voles and dwarf hamsters, two wild species of small rodents kept under simulated natural conditions. It was found that substitution of a LP vivarium for a conventional (non-LP) one doubled the lifespan of female outbred mice, just as SkQ1 did in a non-LP vivarium. SkQ1 prevented age-dependent disappearance of estrous cycles of outbred mice in both LP and non-LP vivaria. In the SPF vivarium in Moscow, male BALB/c mice had shorter lifespan than females, and SkQ1 increased their lifespan to the values of the females. In the females, SkQ1 retarded development of such trait of aging as heart mass increase. Male C57Bl/6 mice housed individually in the SPF vivarium in Stockholm lived as long as females. SkQ1 increased the male lifespan, the longevity of the females being unchanged. SkQ1 did not change food intake by these mice. Dwarf hamsters and mole-voles kept in outdoor cages or under simulated natural conditions lived longer if treated with SkQ1. The effect of SkQ1 on longevity of females is assumed to mainly be due to retardation of the age-linked decline of the immune system. For males under LP or SPF conditions, SkQ1 increased the lifespan, affecting also some other system(s) responsible for aging.