Detcho A. Stoyanovsky

Cytochrome c/cardiolipin relations in mitochondria: a kiss of death

Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. During apoptosis, a mitochondria-specific phospholipid, cardiolipin (CL), interacts with cytochrome c (cyt c) to form a peroxidase complex that catalyzes CL oxidation; this process plays a pivotal role in the mitochondrial stage of the execution of the cell death program. This review is focused on redox mechanisms and essential structural features of cyt cs conversion into a CL-specific peroxidase that represent an interesting and maybe still unique example of a functionally significant ligand change in hemoproteins. Furthermore, specific characteristics of CL in mitochondria--its asymmetric transmembrane distribution and mechanisms of collapse, the regulation of its synthesis, remodeling, and fatty acid composition--are given significant consideration. Finally, new concepts in drug discovery based on the design of mitochondria-targeted inhibitors of cyt c/CL peroxidase and CL peroxidation with antiapoptotic effects are presented.

NITRIC OXIDE ACTIVATES SKELETAL AND CARDIAC RYANODINE RECEPTORS

The endothelial-derived relaxing factor, nitric oxide (NO.) has been shown to depress force in smooth and cardiac muscles through the activation of guanylyl cyclase and an increase in cGMP. In fast skeletal muscle, NO (i.e. NO-related compounds) elicits a modest decrease in developed force, but in contracting muscles NO increases force by a mechanism independent of cGMP. We now demonstrate an alternative mechanism whereby NO triggers Ca2+ release from skeletal and cardiac sarcoplasmic reticulum (SR). NO delivered in the form of NO gas, NONOates (a class of sulfur-free compounds capable of releasing NO), or S-nitrosothiols (R-SNO) oxidized or transnitrosylated regulatory thiols on the release channel (or ryanodine receptor, RyR), resulting in channel opening and Ca2+ release from skeletal and cardiac SR. The process was reversed by sulfhydryl reducing agents which promoted channel closure and Ca2+ reuptake by ATP-driven Ca2+ pumps. NO did not directly alter Ca(2+)-ATPase activity but increased the open probability of RyRs reconstituted in planar bilayers and inhibited [3H]-ryanodine binding to RyRs. The formation of peroxynitrite or thiyl radicals did not account for the reversible R-SNO-dependent activation of RyRs. Ca2+ release induced by nitric oxide free radicals (NO.) was potentiated by cysteine providing compelling evidence that NO. in the presence of O2 formed nitrosylated cysteine followed by the transnitrosation of regulatory thiols on the RyR to activate the channel. These findings demonstrate direct interactions of NO derivatives with RyRs and a new fundamental mechanism to regulate force in striated muscle.

Mitochondrial targeting of electron scavenging antioxidants: Regulation of selective oxidation vs random chain reactions

Effective regulation of highly compartmentalized production of reactive oxygen species and peroxidation reactions in mitochondria requires targeting of small molecule antioxidants and antioxidant enzymes into the organelles. This review describes recently developed approaches to mitochondrial targeting of small biologically active molecules based on: (i) preferential accumulation in mitochondria because of their hydrophobicity and positive charge (hydrophobic cations), (ii) binding with high affinity to an intra-mitochondrial constituent, and (iii) metabolic conversions by specific mitochondrial enzymes to reveal an active entity. In addition, targeted delivery of antioxidant enzymes via expression of leader sequences directing the proteins into mitochondria is considered. Examples of successful antioxidant and anti-apoptotic protection based on the ability of targeted cargoes to inhibit cytochrome c-catalyzed peroxidation of a mitochondria-specific phospholipid cardiolipin, in vitro and in vivo are presented. Particular emphasis is placed on the employment of triphenylphosphonium- and hemi-gramicidin S-moieties as two effective vehicles for mitochondrial delivery of antioxidants.

Endogenous ascorbate regenerates vitamin E in the retina directly and in combination with exogenous dihydrolipoic acid

Vitamin E (alpha-tocopherol) is the major lipid-soluble antioxidant of retinal membranes whose deficiency causes retinal degeneration. Its antioxidant function is realized via scavenging peroxyl radicals as a result of which phenoxyl radicals of alpha-tocopherol are formed. Our hypothesis is that alpha-tocopherol phenoxyl radicals can be reduced by endogenous reductants in the retina, providing for alpha-tocopherol recycling. The results of this study demonstrate for the first time that: (i) endogenous ascorbate (vitamin C) in retinal homogenates and in rod outer segments is able to protect endogenous alpha-tocopherol against oxidation induced by UV-irradiation by reducing the phenoxyl radical of alpha-tocopherol, (ii) in the absence of ascorbate, neither endogenous nor exogenously added glutathione (GSH) is efficient in protecting alpha-tocopherol against oxidation; (iii) GSH does not substantially enhance the protective effect of ascorbate against alpha-tocopherol oxidation; (iv) exogenous dihydrolipoic acid (DHLA), although inefficient in direct reduction of the alpha-tocopherol phenoxyl radical, is able to enhance the protective effect of ascorbate by regenerating it from dehydroascorbate. Thus, regeneration of alpha-tocopherol from its phenoxyl radical can enhance its antioxidant effectiveness in the retina. The recycling of alpha-tocopherol opens new avenues for pharmacological approaches to enhance antioxidants of the retina.

Nitric Oxide Inhibits Peroxidase Activity of Cytochrome c· Cardiolipin Complex and Blocks Cardiolipin Oxidation

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c·CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO–, respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c·CL complex, NO chemically reacted with H2O2-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H2O2-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H2O2-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c·CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3′-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1′2,2′-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1′2,2′-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c·CL complexes.

A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death

The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of hemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids which blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its heme-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 hr before through 24 hrs after the irradiation.

Nitrosative Stress Inhibits the Aminophospholipid Translocase Resulting in Phosphatidylserine Externalization and Macrophage Engulfment IMPLICATIONS FOR THE RESOLUTION OF INFLAMMATION

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of “nitrosatively” modified cells by RAW 264.7 macrophages. Using S-nitroso-l-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation.

A Mitochondria-Targeted Triphenylphosphonium-Conjugated Nitroxide Functions as a Radioprotector/Mitigator

Abstract Jiang, J., Stoyanovsky, D. A., Belikova, N. A., Tyurina, Y. Y., Zhao, Q., Tungekar, M. A., Kapralova, V., Huang, Z., Mintz, A. H., Greenberger, J. S. and Kagan, V. E. A Mitochondria-Targeted Triphenylphosphonium-Conjugated Nitroxide Functions as a Radioprotector/Mitigator. Removal of excessive mitochondrial reactive oxygen species by electron scavengers and antioxidants is a promising therapeutic strategy to reduce the detrimental effects of radiation exposure. Here we exploited triphenylphosphonium (TPP) cation as a means to target nitroxide radicals to mitochondria. We synthesized a library of TPP-conjugated nitroxides and tested their radioprotective effects in γ-irradiated mouse embryo cells and human epithelial BEAS-2B cells. Cells were incubated with conjugates either before or after irradiation. We found that [2-(1-oxyl-2,2,6,6-tetramethyl-piperidin-4-ylimino)-ethyl]-triphenyl-phosphonium (TPEY-Tempo) significantly blocked radiation-induced apoptosis as revealed by externalization of phosphatidylserine on the cell surface and inhibition of cytochrome c release from mitochondria. Using electron paramagnetic resonance, we showed that TPEY-Tempo was integrated into cells and mitochondria, where it underwent one-electron reduction to hydroxylamine. TPEY-Tempo acted as an electron scavenger that prevented superoxide generation and cardiolipin oxidation in mitochondria. Finally, TPEY-Tempo increased the clonogenic survival rate of irradiated cells. The cellular integration efficiencies of nonradioprotective TPP conjugates, including Mito-Tempo (Alexis, San Diego, CA), were markedly lower, although these homologues were integrated into isolated succinate-energized mitochondria to a similar extent as TPEY-Tempo. We conclude that mitochondrial targeting of TPP-conjugated nitroxides represents a promising approach for the development of novel radioprotectors.

Nitric oxide regulates the 26S proteasome in vascular smooth muscle cells

It is well established that nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome is integral to protein degradation and tightly regulates cell cycle proteins. Therefore, we hypothesized that NO directly inhibits the activity of the 26S proteasome. The three enzymatic activities (chymotrypsin-like, trypsin-like and caspase-like) of the 26S proteasome were examined in VSMC. At baseline, caspase-like activity was approximately 3.5-fold greater than chymotrypsin- and trypsin-like activities. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited all three catalytically active sites in a time- and concentration-dependent manner (P<0.05). Caspase-like activity was inhibited to a greater degree (77.2% P<0.05). cGMP and cAMP analogs and inhibitors had no statistically significant effect on basal or NO-mediated inhibition of proteasome activity. Dithiothreitol, a reducing agent, prevented and reversed the NO-mediated inhibition of the 26S proteasome. Nitroso-cysteine analysis following S-nitrosoglutathione exposure revealed that the 20S catalytic core of the 26S proteasome contains 10 cysteines which were S-nitrosylated by NO. Evaluation of 26S proteasome subunit protein expression revealed differential regulation of the alpha and beta subunits in VSMC following exposure to NO. Finally, immunohistochemical analysis of subunit expression revealed distinct intracellular localization of the 26S proteasomal subunits at baseline and confirmed upregulation of distinct subunits following NO exposure. In conclusion, NO reversibly inhibits the catalytic activity of the 26S proteasome through S-nitrosylation and differentially regulates proteasomal subunit expression. This may be one mechanism by which NO exerts its effects on the cell cycle and inhibits cellular proliferation in the vasculature.

Interaction of 1-Hydroxyethyl Radical With Glutathione, Ascorbic Acid and α-Tocopherol

Ethanol has been shown to be oxidized to a free radical metabolite, the 1-hydroxyethyl radical (HER). Interaction of HER with cellular antioxidants may contribute to the known ability of ethanol administration to lower levels of GSH and α-tocopherol. Experiments were carried out to establish a model system for the generation of HER and to study its interaction with GSH, ascorbic acid and α-tocopherol. A standard reaction for formation of azo-compounds using acetaldehyde and hydroxylamine-O-sulfonic acid was applied for the synthesis of 1,1′-dihydroxyazoethane (CH3CH(OH)NNCH(OH)CH3). Although stable at −70°C, thermal decomposition of this compound at room temperature was shown to produce HER, detected by EPR spectrometry as the PBN/HER or DMPO/HER spin adducts, and validated by computer simulation. GSH, present at the beginning of the experiment, inhibited formation of the PBN/HER signal. However, GSH did not cause any decay of pre-formed PBN/HER spin adduct. GSH was consumed in the presence of the HER-generating system in a reaction largely reversed by addition of NADPH plus glutathione reductase. Ascorbate also inhibited formation of the PBN/HER spin adduct and rapidly reduced the pre-formed adduct. HER amplified the oxidation of ascorbate, which was associated with the formation of the semidehydroascorbyl radical. α-Tocopherol was also consumed in the presence of HER. Production of HER in intact HepG2 cells by the redox cycling of 2,3-dimethoxy-1,4-naphthoquinone was associated with consumption of GSH. These data demonstrate the use of a simple chemical system for the controlled, continuous formation of HER and indicate that cellular antioxidants such as GSH, ascorbate, and α-tocopherol, interact with HER. The ability of agents such as ascorbate to reduce the PBN/HER spin adduct to EPR-silent product(s) may mask the quantitative detection of HER in biological systems.