NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of depolarized mitochondria by mitophagy.

Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified as a mitophageal signal recognized by the microtubule-associated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic 'eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2.

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic 'eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.

Molecular design of new inhibitors of peroxidase activity of cytochrome c/cardiolipin complexes: fluorescent oxadiazole-derivatized cardiolipin.

Interaction of a mitochondria-specific anionic phospholipid, cardiolipin (CL), with an intermembrane protein, cytochrome c (cyt c), yields a peroxidase complex. During apoptosis, the complex induces accumulation of CL oxidation products that are essential for detachment of cyt c from the mitochondrial membrane, induction of permeability transition, and release of proapoptotic factors into the cytosol. Therefore, suppression of the peroxidase activity and prevention of CL oxidation may lead to discovery of new antiapoptotic drugs. Here, we report a new approach to regulate the cyt c peroxidase activity by using modified CL with an oxidizable and fluorescent 7-nitro-2,1,3-benzoxadiazole (NBD) moiety (NBD-CL). We demonstrate that NBD-CL forms high-affinity complexes with cyt c and blocks cyt c-catalyzed oxidation of several peroxidase substrates, cyt c self-oxidation, and, most importantly, inhibits cyt c-dependent oxidation of polyunsaturated tetralinoleoyl CL (TLCL) and accumulation of TLCL hydroperoxides. Electrospray ionization mass spectrometry and fluorescence analysis revealed that oxidation and cleavage of the NBD moiety of NBD-CL underlie the inhibition mechanism. We conclude that modified CL combining a nonoxidizable monounsaturated trioleoyl CL with a C(12)-NBD fragment undergoes a regiospecific oxidation thereby representing a novel inhibitor of cyt c peroxidase activity.

The "pro-apoptotic genies" get out of mitochondria: oxidative lipidomics and redox activity of cytochrome c/cardiolipin complexes.

One of the prominent consequences of the symbiogenic origin of eukaryotic cells is the unique presence of one particular class of phospholipids, cardiolipin (CL), in mitochondria. As the product originated from the evolution of symbiotic bacteria, CL is predominantly confined to the inner mitochondrial membrane in normally functioning cells. Recent findings identified CL and its oxidation products as important participants and signaling molecules in the apoptotic cell death program. Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for the interactions with cyt c, one of the major proteins of the intermembrane space. Binding of CL with cytochrome c (cyt c) yields the cyt c/CL complex that acts as a potent CL-specific peroxidase and generates CL hydroperoxides. In this review, we discuss the catalytic mechanisms of CL oxidation by the peroxidase activity of cyt c as well as the role of oxidized CL (CLox) in the release of pro-apoptotic factors from mitochondria into the cytosol. Potential implications of cyt c/CL peroxidase intracellular complexes in disease conditions (cancer, neurodegeneration) are also considered. The discovery of the new role of cyt c/CL complexes in early mitochondrial apoptosis offers interesting opportunities for new targets in drug discovery programs. Finally, exit of cyt c from damaged and/or dying (apoptotic) cells into extracellular compartments and its accumulation in biofluids is discussed in lieu of the formation of its peroxidase complexes with negatively charged lipids and their significance in the development of systemic oxidative stress in circulation.

[Comparative study of the effect of alpha-tocopherol, its synthetic metabolite and ionol on dexamethasone-induced apoptosis in rat thymocytes]. / Sravitel'noe issledovanie deistviia alpha-tokoferola, ego sinteticheskogo metabolita i ionola na indutsirovannyi deksametazonom apoptoz timotsitov krys.

alpha-Tocopherol was found to decrease the level of rat thymocytes DNA fragmentation and to increase the viability of these cells under apoptosis induction by dexamethazone. So the antiapoptotic role of this vitamin is suggested. In contrast to alpha-tocopherol synthetic antioxidant ionol and alpha-tocopherylacetate, contained only 6 carbon atoms in its isoprenoid side chain caused the cell death from necrosis. This process preceded the apoptosis stimulated by dexamethazone.

[Vitamin E and apoptosis]. / Vitamin E i apoptoz.

The literature data concerning the participation of tocopherol in apoptosis are discussed. Acting as antioxidant this vitamin exerts a complex effect on apoptosis mechanisms. Its action on this process is caused by involvement of some different mechanisms transducing the apoptotic signal. Among them are caspase and Fas-receptor activation, sphingosine metabolism, processes carried out in nuclei and mitochondria and signal transduction pathways. The specific mechanisms connected with interaction of this vitamin with tocopherol-binding proteins may be also involved in this vitamin action.

[Effect of alpha-tocopherol on the respiratory burst of neutrophils, blast transformation of lymphocytes, and activity of human natural killer cells in blood]. / Vliianie alpha-tokoferola na dykhatel'nyi vzryv neitrofilov, blasttransformatsiiu limfotsitov i aktivnost' natural'nykh killerov krovi cheloveka.

The effect of vitamin E on immunological reactions of blood cells was studied. The addition of vitamin E in concentration of 100 microM to neutrophils caused the increase of superoxide production. But this index was decreased when incubation of these cells with A23187 or FMLP was accompanied by alpha-tocopherol in concentration of 100 microM followed by the removal of free ligand or alpha-tocopherol in concentration of 0.5 microM. In the presence of PMA the inhibiting action of alpha-tocopherol was not found (under the 0.5 microM of alpha-tocopherol) or was small (under the concentration of it 100 microM of alpha-tocopherol). The addition of alpha-tocopherol in concentration of 0.5 microM and 50 microM caused the inhibition of blasttransformation of lymphocytes and activity of natural killer cells. This effect was expressed more under the low contents of this vitamin in the incubation medium. The level of blasstransformation of blood in vitamin E-deficient rats was by 20% less than in normal. The exogenous addition of alpha-tocopherol in concentration of 0.5 microM did not affect this value. It was suggested that vitamin E can affect the respiration burst of neutrophils, blasttransformation of lymphocytes and activity of natural killer cells and these actions depend on its concentrations.

[Role of Ca ions and protein kinase C in the action of vitamin E on respiratory burst of neutrophils and blast transformation of lymphocytes]. / Uchastie ionov Ca i proteinkinazy C v deistvii vitamina E na dykhatel'nyi vzryv neitrofilov i blasttransformatsiiu limfotsitov.

It was shown that vitamin E decreased the stimulating role of oxidative burst activators which influenced on Ca(2+)-dependent mechanisms (A23187, verapamil, FMLP). Jointly addition of this vitamin and blood plasma contained tocopherol-binding proteins influenced on mechanisms associated with protein kinase C. It was shown that Ca ions can also take part in tocopherol's action on blast transformation of lymphocytes.

Tocopherol modulates the effects of A23187, verapamil, and phorbol myristate acetate on RNA-polymerase activity of isolated rat liver nuclei.

Preincubation of rat liver nuclei with tocopherol decreased inhibition of RNA-polymerase activity of isolated rat liver nuclei by A23187, verapamil, and phorbol myristate acetate (PMA). In nuclei of vitamin E-deficient rats, A23187, verapamil, and PMA did not inhibit label incorporation into RNA with and without tocopherol. A23187, verapamil, and PMA did not inhibit the activity of nuclei treated with 1% Triton X-100; tocopherol activated RNA synthesis but co-administration of A23187 or verapamil and tocopherol had no effect and the combination of tocopherol and PMA inhibited RNA-polymerase activity by 25%. The effect of the combination of verapamil and PMA in the presence of free tocopherol was different from the effect of these compounds assayed in the presence of the complex of tocopherol with tocopherol-binding protein.

[Effect of ionophore A23187 and verapamil on RNA and DNA polymerase activity in rat liver nuclei]. / Vliianie ionofora A23187 i verapamila na RNK- i DNK-polimeraznuiu aktivnost' v iadrakh kletok pecheni krys.

It was shown that verapamil and Ca(2+)-ionofor A23187 essentially inhibited the activity of RNA-polymerase in isolated nuclei of rat liver, while DNA-polymerasing activity was inhibited only after the addition of verapamil. These phenomena were not found after treatment of nuclei with 1% triton X-100. The inhibition of RNA synthesis was also found after the addition of Ca(2+)-ionofor A23187 but not verapamil to nuclei which envelope was disrupted by freezing-thawing procedure. The data obtained suggest that Ca(2+)-ionofor A23187 and verapamil affected RNA and DNA synthesis in isolated nuclei.

[Effect of alpha-tocopherol and nuclear tocopherol-binding proteins on DNA-polymerase activity of isolated nuclei and nuclear matrix]. / Vliianie alpha-tokoferola i iadernykh tokoferolsviazyvaiushchikh belkov na DNK-polimeraznuiu aktivnost' izolirovannykh iader i iadernogo matriksa.

It was shown, that tocopherol addition to the incubation medium increased the DNA-polymerase activity of isolated rat liver nuclei. This phenomenon was not discovered in nuclei of vitamin E-depleted rats. The action of tocopherol changed after the extraction of nuclei with triton X-100. In this situation tocopherol decreased DNA-polymerase activity of nuclei. The differences between rates of DNA synthesis was found after simultaneous addition of tocopherol and fraction of tocopherol-binding protein to the nuclei of normal and vitamin E-depleted rats. In this case DNA-polymerase activity of normal nuclei increased, but did not change in nuclei of vitamin E undernourished rats. The difference between them was about 30%. The differences between DNA-polymerase activity of normal and vitamin E-depleted rats after the addition of tocopherol and fraction of tocopherol-binding protein were also found in nuclear matrix preparation. It was supposed that tocopherol was able to take part in functioning of cell nuclei modulating DNA synthesis as well.

[Role of S-100 protein in the function of brain cell nuclei]. / Rol' belka S-100 v funktsionirovanii kletochnykh iader mozga.

Data on physico-chemical properties and functional role of protein S-100 have been generalized. An analysis of literary data permits making a conclusion that the protein interaction with Ca2+ ions plays great part in its action mechanisms. Data are presented on the properties of the family of Ca-binding proteins similar to protein S-100 as to their structure. Peculiar attention is paid to the analysis of this protein function in the cellular nucleus, changing the degree of proteins phosphorylation and RNA synthesis.

[Effect of alpha-tocopherol and ubiquinone on mitochondrial RNA polymerase activity. The role of tocopherol-binding proteins]. / Vliianie alpha-tokoferola i ubikhinona na RNK-polimeraznuiu aktivnost' mitokhondrii. Rol' tokoferolsviazyvaiushchikh belkov.

It has been shown that alpha-tocopherol within the complex with tocopherol-binding proteins, in contrast with free tocopherol, decreases the RNA-polymerase activity in isolated rat liver mitochondria. Both free and complexed to proteins alpha-tocopherol increases the RNA-polymerase activity in isolated nuclei. The effect of alpha-tocopherol is conditioned mostly by the nature of the tocopherol-binding proteins. It has been established that ubiquinone complexed to tocopherol-binding proteins from mitochondria can also change the level of RNA synthesis. The effect of alpha-tocopherol and ubiquinone are not additive.

[Chromatin proteins binding vitamin E]. / Issledovanie belkov khromatina, sviazyvaiushchikh vitamin E.

It was discovered that alpha-tocopherol binding with isolated chromatin is specific only when fraction of tocopherol-binding proteins from a nuclear extract with 1% triton X-100 is present. During the chromatography of chromatin incubated with [H] alpha-tocopherol on hydroxyapatite a specific binding activity was present only in fraction eluted with 2M NaCl + 5M urea. Quantitative changes in the protein content of this fraction during a hypovitaminosis are found. It is shown that vitamin E can effect the RNA polymerase activity of a nuclear matrix in the in vitro assays. It is suggested that the presence of tocopherol-binding proteins of chromatin in necessary for such an action of alpha-tocopherol.

[Effect of brain cytosol proteins of embryonal and adult animals on RNA-polymerase activity of isolated brain nuclei]. / Vliianie belkov tsitozolia mozga émbrionov i vroslykh zhivotnykh na RNK-polimeraznuiu aktivnost' izolirovannykh iader mozga.

Cytosol and its fractions obtained by the precipitation with ammonium sulphate and ion-exchange chromatography have been studied for their effect on the RNA-polymerase activity of isolated nuclei. We observed the discrepancies in the action of total cytosol of embryonal, newborn or adult animals on the label's incorporation in RNA. It was found that some fractions increased DNA-polymerase activity of isolated nuclei in cattle embryonal cytosol. The same fractions obtained from adult cytosol did not act in such a way. It was found that most fractions obtained from cytosol of adult brain inhibited the RNA-polymerase activity of brain nuclei.

[Incorporation of [3H]alpha-tocopherol into isolated nuclei and its binding by rat liver chromatin]. / Vkliuchenie [3H]alpha-tokoferola v izolirovannye iadra i ego sviazyvanie s khromatinom pecheni krys.

The investigation of inclusion of [3H]alpha-tocopherol to isolated rat liver nuclei has revealed its nonspecific character. The presence of cytosol is necessary for specific interaction of alpha-tocopherol with nuclei. After the centrifugation of preliminarily labeled chromatin the most quantity of tocopherol was bound with oligonucleosomes and pelleted chromatin. It is supposed, that the preservation of supernucleosomes level of chromatin folding was necessary for the interaction of alpha-tocopherol with chromatin.

[Effect of vitamin E on transcription in isolated nuclei and rat liver chromatin in normal status and in E-hypovitaminosis]. / Vliianie vitamina E na transkriptsiiu v izolirovannykh iadriakh i khromatine pecheni krys v norme i pri E-gipovitaminoze.

The effect of alpha-tocopherol on the RNA-polymerase activity in isolated rat nuclei and chromatin from normal and E-deficient rats and the possible role of tocopherol-binding proteins in this process were studied. Some differences in the RNA-polymerase activities of the nuclei were found; however, in vitro added alpha-tocopherol had no effect on the level of the label incorporation into RNA. No effect of alpha-tocopherol on this process was observed after addition of cytosol either. Analysis of chromatins from normal and E-deficient rats revealed no differences in their RNA-polymerase activities. In vitro added alpha-tocopherol increased the RNA-polymerase activity of normal (but not of vitamin E-deficient) rats. Some differences in the RNA-polymerase activities were noted after addition to the incubation medium of the Triton X-100-solubilized nuclear fraction specifically binding alpha-tocopherol. This effect was enhanced in the presence of exogenous alpha-tocopherol. The susceptibility of chromatin from normal and E-deficient rats to DNAse I hydrolysis was also found to be different. It was concluded that vitamin E can influence the RNA-polymerase activity of the nuclei and chromatin as well as the chromatin structure and that alpha-tocopherol-binding proteins are necessary for the vitamin E effect on the RNA-polymerase activity to be manifested.

[Changes in protein, lipid composition, DNA- and RNA- polymerase activity of the chromatin fraction and the nuclear matrix of the rat liver in hypovitaminosis E]. / Izmeneniia belkovogo, lipidnogo sostava, DNK- i RNK-polimeraznoi aktivnosti fraktsii khromatina i iadernogo matriksa pecheni krys v usloviiakh E-gipovitaminoza.

E-hypovitaminosis-induced antioxidant deficiency in rats causes changes in some properties of nuclear structures of the liver cells, i.e. fractions of transcriptionally active and repressed chromatin and nuclear matrix. Changes are found in the protein spectrum of the fraction of transcriptionally active chromatin and nuclear matrix. Lipids of transcriptionally active and repressed chromatin fractions may be peroxidated when this process is stimulated in the NADPH- and ascorbate-dependent systems. In antioxidant deficiency these processes are intensified in the fractions of repressed chromatin. E-hypovitaminosis leads to changes in the fatty acid spectrum of chromatin fractions which correlated with the shifts in the process of lipid peroxidation. Antioxidant deficiency produces changes in the activities of endogenous DNA- and RNA-polymerases in chromatin fractions and in the nuclear matrix. In the fraction of the transcriptionally active liver chromatin of E-deficient animals the endogenous total DNA-polymerase activity and the activity of DNA-polymerases alpha and beta decrease, while in the fractions of repressed chromatin the total RNA-polymerase activity increases. In E-hypovitaminosis the endogenous DNA- and RNA-polymerase activities in the nuclear matrix decrease. Addition of alpha-tocopherol to the preparations of the isolated nuclear matrix results in an increase of the DNA- and RNA-polymerase activities which is more vivid in preparations made of the E-hypovitaminous animal liver.

[Tocopherol-binding proteins in membranes of rat liver mitochondria]. / Issledovanie tokoferolsviazyvaiushchikh belkov membran mitokhondrii pecheni krys.

The use of the adsorption chromatography on the hydroxyl apatite makes it possible to yield and partly purify the triton X-100-solubilized mitochondrial protein fraction of the rat liver able to bind specifically [3H]-alpha-tocopherol. The method permits removing simultaneously both free detergent and [3H]-alpha-tocopherol from the protein mixture without disturbance of the established equilibrium. When compared with methods used for the removal of free hydrophobic ligands in the in vitro binding experiments, the applied method is the most effective.