RESUMEN
Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration-dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at the sn-2 position, generating the corresponding PUFA-lyso PtdCho. This specificity for PUFA-PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species.
Asunto(s)
Lipasa/antagonistas & inhibidores , Lipoproteínas/metabolismo , Esfingomielinas/farmacología , Humanos , Hidrólisis , Técnicas In Vitro , Lipoproteínas/química , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Liposomas , Fosfatidilcolinas/metabolismo , Proteínas Recombinantes/metabolismo , Esfingomielinas/metabolismo , Especificidad por SustratoRESUMEN
Although marine oysters contain abundant amounts of ether-linked aminophospholipids, the structural identification of the various molecular species has not been reported. We developed a normal-phase silica liquid chromatography/negative-ion electrospray ionization/quadrupole multiple-stage linear ion-trap mass spectrometric (NPLC-NI-ESI/Q-TRAP-MS(3)) method for the structural elucidation of ether molecular species of serine and ethanolamine phospholipids from marine oysters. The major advantages of the approach are (i) to avoid incorrect selection of isobaric precursor ions derived from different phospholipid classes in a lipid mixture, and to generate informative and clear MS(n) product ion mass spectra of the species for the identification of the sn-1 plasmanyl or plasmenyl linkages, and (ii) to increase precursor ion intensities by "concentrating" lipid molecules of each phospholipid class for further structural determination of minor molecular species. Employing a combination of NPLC-NI-ESI/MS(3) and NPLC-NI-ESI/MS(2), we elucidated, for the first time, the chemical structures of docosahexaenoyl and eicosapentaenoyl plasmenyl phosphatidylserine (PS) species and differentiated up to six isobaric species of diacyl/alkylacyl/alkenylacyl phosphatidylethanolamine (PE) in the US pacific oysters. The presence of a high content of both omega-3 plasmenyl PS/plasmenyl PE species and multiple isobaric molecular species isomers is the noteworthy characteristic of the marine oyster. The simple and robust NPLC-NI-ESI/MS(n)-based methodology should be particularly valuable in the detailed characterization of marine lipid dietary supplements with respect to omega-3 aminophospholipids.
Asunto(s)
Ostreidae/química , Éteres Fosfolípidos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Cromatografía Liquida/economía , Cromatografía Liquida/métodos , Suplementos Dietéticos/análisis , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/aislamiento & purificación , Fosfatidilserinas/química , Fosfatidilserinas/aislamiento & purificación , Éteres Fosfolípidos/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray/economíaRESUMEN
Although sphingomyelin (SM) is the most abundant phospholipid in the plasma, next to phosphatidylcholine (PC), its physiological function in plasma is unclear. Here we employed plasma from various genetic models of mice which naturally differ in their plasma SM/PC ratios, to study the role of SM as a modulator of LCAT, the enzyme responsible for HDL maturation and the synthesis of cholesteryl esters (CE) in normal plasma. Serine palmitoyltransferase deficient mice, and SM synthase deficient mice, both of which have below normal SM/PC ratios, showed significantly elevated LCAT activities when assayed with the endogenous substrates. On the other hand, LDL receptor knockout mice, and apo E knockout mice, both of which have high SM/PC ratios, had markedly reduced (-80%) LCAT activities. The LCAT levels in plasma, as assayed with an exogenous substrate, were similar in all groups, except for a 45% decrease in apo E knockout mice. Plasma samples with high SM/PC ratios had lower percentage of 20:4, 22:5, and 22:6 CE all of which are formed by LCAT, and a higher percentage of the atherogenic 18:1 CE which is mainly derived from the action of liver ACAT, showing that in vivo, the contribution of LCAT to plasma CE is reduced while that of liver ACAT is increased. These results show that SM is a physiological modulator of LCAT activity as well as plasma CE composition, and this may contribute to the previously reported pro-atherogenic effect of high plasma SM levels.
Asunto(s)
Ésteres del Colesterol/metabolismo , Colesterol/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Esfingomielinas/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Colesterol/sangre , Ésteres del Colesterol/sangre , Esterificación , Lípidos/sangre , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolinas/sangre , Fosfatidilcolinas/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de LDL/metabolismo , Serina C-Palmitoiltransferasa/deficiencia , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/metabolismo , Esfingomielinas/sangre , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/deficiencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
Probucol inhibits the proliferation of vascular smooth muscle cells in vitro and in vivo, and the drug reduces intimal hyperplasia and atherosclerosis in animals via induction of heme oxygenase-1 (HO-1). Because the succinyl ester of probucol, succinobucol, recently failed as an antiatherogenic drug in humans, we investigated its effects on smooth muscle cell proliferation. Succinobucol and probucol induced HO-1 and decreased cell proliferation in rat aortic smooth muscle cells. However, whereas inhibition of HO-1 reversed the antiproliferative effects of probucol, this was not observed with succinobucol. Instead, succinobucol but not probucol induced caspase activity and apoptosis, and it increased mitochondrial oxidation of hydroethidine to ethidium, suggestive of the participation of H(2)O(2) and cytochrome c. Also, succinobucol but not probucol converted cytochrome c into a peroxidase in the presence of H(2)O(2), and succinobucol-induced apoptosis was decreased in cells that lacked cytochrome c or a functional mitochondrial complex II. In addition, succinobucol increased apoptosis of vascular smooth muscle cells in vivo after balloon angioplasty-mediated vascular injury. Our results suggest that succinobucol induces apoptosis via a pathway involving mitochondrial complex II, H(2)O(2), and cytochrome c. These unexpected results are discussed in light of the failure of succinobucol as an antiatherogenic drug in humans.
Asunto(s)
Apoptosis/efectos de los fármacos , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/fisiología , Probucol/análogos & derivados , Animales , Aorta/citología , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocromos c/metabolismo , Fragmentación del ADN , Complejo II de Transporte de Electrones/metabolismo , Inducción Enzimática/efectos de los fármacos , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Peróxido de Hidrógeno/metabolismo , Masculino , Metaloporfirinas/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Probucol/farmacología , Protoporfirinas/farmacología , Conejos , RatasRESUMEN
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 haemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids that blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its haem-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 h before through 24 h after the irradiation.
Asunto(s)
Muerte Celular/efectos de los fármacos , Citocromo-c Peroxidasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Mitocondrias/efectos de los fármacos , Protectores contra Radiación/farmacología , Animales , Muerte Celular/efectos de la radiación , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/química , Femenino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/enzimología , Modelos Moleculares , Simulación de Dinámica Molecular , Protectores contra Radiación/químicaRESUMEN
Ascorbate is a vital reductant/free radical scavenger in the CNS, whose content defines - to a large extent - the redox status and the antioxidant reserves. Quick, reliable and specific methods for its measurement in brain samples are highly desirable. We have developed a new high-throughput screening assay for measurements of ascorbate using a fluorescence plate-reader. This assay is based on a direct reaction of ascorbate with a nitroxide radical conjugated with a fluorogenic acridine moiety, 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl radical (AC-TEMPO), yielding fluorescent hydroxylamine product (AC-TEMPO-H). The reaction was monitored over time using fluorescence and electron spin resonance techniques. The appearance of fluorescent AC-TEMPO-H was linear within the range of 3.75-75µM AscH(-) in the sample (0.5-10µM AscH(-) in the well). Assay was validated with high performance liquid chromatography method. The concentration of ascorbate in murine tissue samples, including brain samples after traumatic brain injury and hemorrhagic shock, was measured.
Asunto(s)
Ácido Ascórbico/química , Química Encefálica , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Ácido Ascórbico/fisiología , Química Encefálica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Ionizing radiation triggers mitochondrial overproduction of H(2)O(2) with concomitant induction of intrinsic apoptosis, whereby clearance of H(2)O(2) upon overexpression of mitochondrial catalase increases radioresistance in vitro and in vivo. As an alternative to gene therapy, we tested the potential of Mn((III))-porphyrin complexes to clear mitochondrial H(2)O(2). We report that triphenyl-[(2E)-2-[4-[(1Z,4Z,9Z,15Z)-10,15,20-tris(4-aminophenyl)-21,23-dihydroporphyrin-5-yl]phenyl]iminoethyl]phosphonium-Mn((III)) compartmentalizes preferentially into mitochondria of mouse embryonic cells, reacts with H(2)O(2), impedes γ-ray-induced mitochondrial apoptosis, and increases the survival of mice exposed to whole body irradiation with γ-rays.
RESUMEN
Glioblastoma multiforme is the most frequent and aggressive primary brain tumor. A strong rationale to identify innovative approaches to treat these tumors is required since treatment failures result in local recurrences and median survivals range from 9 to 12 months. Glioma cells are reported to have less mitochondrial content compared to adjacent normal brain cells. Based on this difference, we suggest a new strategy, utilizing protection of normal brain cells by mitochondria-targeted electron scavengers and antioxidants-nitroxides-thus allowing for the escalation of the radiation doses. In this paper, we report that a conjugate of nitroxide with a hydrophobic cation, triphenyl-phosphonium (TPEY-Tempo), significantly protected brain endothelial cells from γ-irradiation-induced apoptosis while radiosensitizing brain tumor cells. Thus, TPEY-Tempo may be a promising adjunct in the treatment of glioblastoma with the potential to not only prolong survival but also to maintain quality of life and reduce treatment toxicity.
Asunto(s)
Apoptosis/efectos de los fármacos , Encéfalo/citología , Mitocondrias/efectos de la radiación , Fármacos Neuroprotectores/farmacología , Óxidos de Nitrógeno/química , Compuestos de Organoselenio/farmacología , Apoptosis/efectos de la radiación , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/ultraestructura , Caspasa 3/metabolismo , Línea Celular Transformada , Óxidos N-Cíclicos/metabolismo , Citocromos c/metabolismo , Relación Dosis-Respuesta en la Radiación , Complejo IV de Transporte de Electrones/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/efectos de la radiación , Células Endoteliales/ultraestructura , Rayos gamma/efectos adversos , Glioma/patología , Glioma/ultraestructura , Humanos , Mitocondrias/metabolismo , Compuestos Orgánicos/metabolismoRESUMEN
Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.
Asunto(s)
Citocromos c/metabolismo , Mutación , Organofosfatos/química , Tirosina/metabolismo , Animales , Apoptosis , Cardiolipinas/metabolismo , Caspasas/metabolismo , Bovinos , Respiración de la Célula , Citocromos c/química , Citocromos c/genética , Ratones , Fosforilación , Unión Proteica , RatasRESUMEN
We have shown previously that single-walled carbon nanotubes can be catalytically biodegraded over several weeks by the plant-derived enzyme, horseradish peroxidase. However, whether peroxidase intermediates generated inside human cells or biofluids are involved in the biodegradation of carbon nanotubes has not been explored. Here, we show that hypochlorite and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase catalyse the biodegradation of single-walled carbon nanotubes in vitro, in neutrophils and to a lesser degree in macrophages. Molecular modelling suggests that interactions of basic amino acids of the enzyme with the carboxyls on the carbon nanotubes position the nanotubes near the catalytic site. Importantly, the biodegraded nanotubes do not generate an inflammatory response when aspirated into the lungs of mice. Our findings suggest that the extent to which carbon nanotubes are biodegraded may be a major determinant of the scale and severity of the associated inflammatory responses in exposed individuals.
Asunto(s)
Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidad , Neutrófilos/enzimología , Peroxidasa/metabolismo , Neumonía/inducido químicamente , Neumonía/patología , Animales , Humanos , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Nanotubos de Carbono/ultraestructura , Neutrófilos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Espectrofotometría Infrarroja , Espectrometría RamanRESUMEN
Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of ischemic injury, but the exact mechanisms responsible for its toxicity remain unclear. Infection of primary neurons with an adenovirus expressing wild type (WT) COX-2 increased the susceptibility of neurons to hypoxia. Infection with an adenoviral vector expressing COX-2 with a mutation at the cyclooxygenase site did not increase susceptibility to hypoxia, whereas over-expression of COX-2 with a mutation in the peroxidase site produced similar susceptibility to hypoxia as WT COX-2. Primary neuronal cultures obtained from transgenic mice bearing a mutation in the COX-2 cylooxygenase site were protected from hypoxia. Mice with a mutation in the cyclooxygenase site had smaller infarctions 24 h after 70 min of middle cerebral artery occlusion than WT control mice. COX-2 activity had no effect on the formation of protein carbonyls. Ascorbate radicals were detected by electron paramagnetic resonance as a product of recombinant COX-2 activity and were blocked by COX-2 inhibitors. Similarly, formation of ascorbate radicals was inhibited in the presence of COX-2 inhibitors and in homogenates obtained from COX-2 null mice. Taken together, these results indicate that the cyclooxygenase activity of COX-2 is necessary to exacerbate neuronal hypoxia/ischemia injury rather than the peroxidase activity of the enzyme.
Asunto(s)
Infarto Encefálico/enzimología , Ciclooxigenasa 2/metabolismo , Hipoxia-Isquemia Encefálica/enzimología , Degeneración Nerviosa/enzimología , Animales , Ácido Araquidónico/metabolismo , Ácido Ascórbico/metabolismo , Infarto Encefálico/genética , Infarto Encefálico/fisiopatología , Dominio Catalítico/fisiología , Línea Celular , Células Cultivadas , Ciclooxigenasa 2/química , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa 2/farmacología , Radicales Libres/metabolismo , Humanos , Hipoxia-Isquemia Encefálica/genética , Hipoxia-Isquemia Encefálica/fisiopatología , Ratones , Ratones Transgénicos , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Estrés Oxidativo/fisiología , Peroxidasa/metabolismo , Prostaglandina H2/biosíntesis , RatasRESUMEN
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 gamma-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.
Asunto(s)
Mitocondrias/metabolismo , Óxidos de Nitrógeno/química , Compuestos de Organoselenio/química , Protectores contra Radiación/química , Animales , Western Blotting , Células Cultivadas , Cromatografía Liquida , Ratones , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Manganese superoxide dismutase (MnSOD) is vital to the protection of mitochondria and cells against oxidative stress. Earlier, we demonstrated that catalytically active homo-tetramer of MnSOD can be stabilized by oxidative cross-linking. Here we report that this effect may be translated into increased radioresistance of mouse embryonic cells (MECs) by pre-exposure to oxidative stress. Pre-treatment of MECs with antimycin A, rotenone or H(2)O(2) increased their survival after irradiation. Using MnSOD siRNA, we show that MECs with decreased MnSOD levels displayed a lowered ability to preconditioning. Thus oxidative preconditioning may be used for targeted regulation of MnSOD.
Asunto(s)
Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Superóxido Dismutasa/metabolismo , Animales , Antimicina A/farmacología , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Embrión de Mamíferos/citología , Ratones , Especies Reactivas de Oxígeno/farmacología , Rotenona/farmacología , Factores de TiempoRESUMEN
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.
Asunto(s)
Antioxidantes/farmacología , Sistemas de Liberación de Medicamentos/métodos , Depuradores de Radicales Libres/química , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , Depuradores de Radicales Libres/farmacocinética , Humanos , Mitocondrias/efectos de los fármacos , Modelos Biológicos , Estructura Molecular , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Cytochrome c (cyt c), a mitochondrial intermembrane electron shuttle between complexes III and IV, can, upon binding with an anionic phospholipid, cardiolipin (CL), act as a peroxidase that catalyzes cardiolipin oxidation. H(2)O(2) was considered as a source of oxidative equivalents for this reaction, which is essential for programmed cell death. Here we report that peroxidase cyt c/CL complexes can utilize free fatty acid hydroperoxides (FFA-OOH) at exceptionally high rates that are approximately 3 orders of magnitude higher than for H(2)O(2). Similarly, peroxidase activity of murine liver mitochondria was high with FFA-OOH. Using EPR spin trapping and LC-MS techniques, we have demonstrated that cyt c/CL complexes split FFA-OOH predominantly via a heterolytic mechanism, yielding hydroxy-fatty acids, whereas H(2)O(2) (and tert-butyl hydroperoxide, t-BuOOH) undergo homolytic cleavage. Computer simulations have revealed that Arg(38) and His(33) are important for the heterolytic mechanism at potential FFA-OOH binding sites of cyt c (but not for H(2)O(2) or t-BuOOH). Regulation of FFA-OOH metabolism may be an important function of cyt c that is associated with elimination of toxic FFA-OOH and synthesis of physiologically active hydroxy-fatty acids in mitochondria.
Asunto(s)
Antioxidantes/metabolismo , Cardiolipinas/metabolismo , Citocromos c/metabolismo , Ácidos Grasos/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias Hepáticas/enzimología , Animales , Armoracia/enzimología , Modelos Moleculares , Murinae , Oxidación-Reducción , Unión ProteicaRESUMEN
Generation of reactive oxygen species by damaged respiratory chain followed by the formation of cytochrome c (cyt c)-cardiolipin (CL) complex with peroxidase activity are early events in apoptosis. By quenching the peroxidase activity of cyt c-CL complexes in mitochondria, nitric oxide can exert anti-apoptotic effects. Therefore, mitochondria-targeted pro-drugs capable of gradual nitric oxide radical (NO*) release are promising radioprotectants. Here we demonstrate that (2-hydroxyamino-vinyl)-triphenyl-phosphonium effectively accumulates in mitochondria, releases NO* upon mitochondrial peroxidase reaction, protects mouse embryonic cells from irradiation-induced apoptosis and increases their clonogenic survival after irradiation. We conclude that mitochondria-targeted peroxidase-activatable NO-donors represent a new interesting class of radioprotectors.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Hidroxilaminas/farmacología , Óxido Nítrico/metabolismo , Compuestos Organofosforados/farmacología , Animales , Apoptosis/efectos de la radiación , Biotransformación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Rayos gamma , Hidroxilaminas/farmacocinética , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Donantes de Óxido Nítrico/farmacocinética , Donantes de Óxido Nítrico/farmacología , Compuestos Organofosforados/farmacocinética , Protectores contra Radiación/farmacocinética , Protectores contra Radiación/farmacologíaRESUMEN
Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that alpha-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H(2)O(2). Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage.
Asunto(s)
Citocromos c/metabolismo , Enfermedad de Parkinson/fisiopatología , Peroxidasas/metabolismo , Sinucleínas/metabolismo , Animales , Apoptosis , Cardiolipinas/fisiología , Línea Celular Tumoral , Clonación Molecular , Reactivos de Enlaces Cruzados , Células HeLa/fisiología , Humanos , Lípidos/fisiología , Ratones , Neuroblastoma , Neuronas/fisiología , Estrés Oxidativo , Enfermedad de Parkinson/enzimología , ARN Interferente Pequeño/genética , Sinucleínas/genéticaRESUMEN
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 c's 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.
Asunto(s)
Cardiolipinas/fisiología , Citocromos c/fisiología , Mitocondrias/fisiología , Animales , Apoptosis , Humanos , Peroxidación de Lípido/fisiología , Estrés Oxidativo , Unión Proteica , Transducción de SeñalRESUMEN
Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid "eat-me" signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells.
Asunto(s)
Nanotubos de Carbono/química , Fagocitos/metabolismo , Fosfatidilserinas/química , Animales , Línea Celular Tumoral , Femenino , Citometría de Flujo , Células HeLa , Humanos , Técnicas In Vitro , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Nanotubos de Carbono/toxicidad , Fosfatidilserinas/metabolismo , RatasRESUMEN
The critical role of mitochondria in programmed cell death leads to the design of mitochondriotropic agents as a strategy in regulating apoptosis. For anticancer therapy, stimulation of proapoptotic mitochondrial events in tumor cells and their suppression in surrounding normal cells represents a promising paradigm for new therapies. Different approaches targeting regulation of components of mitochondrial antioxidant system such as Mn-SOD demonstrated significant antitumor efficiency, particularly in combination therapy. This review is focused on a newly discovered early stage of mitochondria-dependent apoptosis - oxidative lipid signaling involving a mitochondria-specific phospholipid cardiolipin (CL). Cytochrome c (cyt c) acts as a CL-specific peroxidase very early in apoptosis. At this stage, the hostile events are still secluded within the mitochondria and do not reach the cytosolic targets. CL oxidation process is required for the release of pro-apoptotic factors into the cytosol. Manipulation of cyt c interactions with CL, inhibition of peroxidase activity, and prevention of CL peroxidation are prime targets for the discovery of anti-apoptotic drugs acting before the "point-of-no-return" in the fulfillment of the cell death program. Therefore, mitochondria-targeted disruptors and inhibitors of cyt c/CL peroxidase complexes and suppression of CL peroxidation represent new strategies in anti-apoptotic drug discovery.