Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.
2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in "diet pills", despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP's uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP's protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.
- MeSH
- 2,4-dinitrofenol farmakologie MeSH
- jaterní mitochondrie metabolismus MeSH
- krysa rodu rattus MeSH
- lipidové dvojvrstvy metabolismus MeSH
- membránové potenciály účinky léků MeSH
- mitochondriální ADP/ATP-translokasy metabolismus MeSH
- mitochondriální odpřahující proteiny metabolismus MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Quercetin and dehydrosilybin are polyphenols which are known to behave like uncouplers of respiration in isolated mitochondria. Here we investigated whether the effect is conserved in whole cells. Following short term incubation, neither compound uncouples mitochondrial respiration in whole H9c2 cells below 50μM. However, following hypoxia, or long term incubation, leak (state IV with oligomycin) oxygen consumption is increased by quercetin. Both compounds partially protected complex I respiration, but not complex II in H9c2 cells following hypoxia. In a permeabilised H9c2 cell model, the increase in leak respiration caused by quercetin is lowered by increased [ADP] and is increased by adenine nucleotide transporter inhibitor, atractyloside, but not bongkrekic acid. Both quercetin and dehydrosilybin dissipate mitochondrial membrane potential in whole cells. In the case of quercetin, the effect is potentiated post hypoxia. Genetically encoded Ca++ sensors, targeted to the mitochondria, enabled the use of fluorescence microscopy to show that quercetin decreased mitochondrial [Ca++] while dehydrosilybin did not. Likewise, quercetin decreases accumulation of [Ca++] in mitochondria following hypoxia. Fluorescent probes were used to show that both compounds decrease plasma membrane potential and increase cytosolic [Ca++]. We conclude that the uncoupler-like effects of these polyphenols are attenuated in whole cells compared to isolated mitochondria, but downstream effects are nevertheless apparent. Results suggest that the effect of quercetin observed in whole and permeabilised cells may originate in the mitochondria, while the mechanism of action of cardioprotection by dehydrosilybin may be less dependent on mitochondrial uncoupling than originally thought. Rather, protective effects may originate due to interactions at the plasma membrane.
- MeSH
- buněčné linie MeSH
- digitonin farmakologie MeSH
- fluorescenční mikroskopie MeSH
- konfokální mikroskopie MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mitochondriální ADP/ATP-translokasy metabolismus MeSH
- quercetin farmakologie MeSH
- silymarin farmakologie MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (PiC) are supposed to form a supercomplex called ATP synthasome. Our protein and transcript analysis of rat tissues indicates that the expression of ANT and PiC is transcriptionally controlled in accordance with the biogenesis of ATP synthase. In contrast, the content of ANT and PiC is increased in ATP synthase deficient patients' fibroblasts, likely due to a post-transcriptional adaptive mechanism. A structural analysis of rat heart mitochondria by immunoprecipitation, blue native/SDS electrophoresis, immunodetection and MS analysis revealed the presence of ATP synthasome. However, the majority of PiC and especially ANT did not associate with ATP synthase, suggesting that most of PiC, ANT and ATP synthase exist as separate entities.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- fibroblasty metabolismus MeSH
- fosfáty chemie metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- makromolekulární látky chemie metabolismus MeSH
- mitochondriální ADP/ATP-translokasy chemie genetika metabolismus MeSH
- mitochondriální protonové ATPasy chemie genetika metabolismus MeSH
- mitochondrie metabolismus MeSH
- novorozená zvířata MeSH
- potkani Wistar MeSH
- srdeční mitochondrie metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei, a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III+IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote.
- MeSH
- mitochondriální ADP/ATP-translokasy chemie genetika metabolismus MeSH
- molekulární evoluce * MeSH
- oxidativní fosforylace * MeSH
- protozoální proteiny chemie genetika metabolismus MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- aktivní transport fyziologie genetika MeSH
- dyslipidemie genetika metabolismus MeSH
- inzulinová rezistence genetika MeSH
- mastné kyseliny metabolismus MeSH
- mitochondriální ADP/ATP-translokasy genetika metabolismus MeSH
- modely u zvířat MeSH
- potkani transgenní MeSH
- regulace genové exprese MeSH
- triglyceridy krev metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Homeostasis of reactive oxygen species (ROS) in cardiomyocytes is critical for elucidation of normal heart physiology and pathology. Mitochondrial phospholipases A2 (mt-PLA2) have been previously suggested to be activated by ROS. Therefore, we have attempted to elucidate physiological role of such activation. We have found that function of a specific i-isoform of mitochondrial phospholipase A2 (mt-iPLA2) is activated by tert-butylhydroperoxide in isolated rat heart mitochondria. Isoform specificity was judged from the inhibition by bromoenol lactone (BEL), a specific iPLA2 inhibitor. Concomitant uncoupling has been caused by free fatty acids, since it was inhibited by bovine serum albumin. The uncoupling was manifested as a respiration burst accompanied by a slight decrease in mitochondrial inner membrane potential. Since this uncoupling was sensitive to carboxyatractyloside and purine nucleotide di- and triphosphates, we conclude that it originated from the onset of fatty acid cycling mediated by the adenine nucleotide translocase (major contribution) and mitochondrial uncoupling protein(s) (minor contribution), respectively. Such a mild uncoupling may provide a feedback downregulation of oxidative stress, since it can further attenuate mitochondrial production of ROS. In conclusion, ROS-induced function of cardiac mt-iPLA2 may stand on a pro-survival side of ischemia-reperfusion injury.
- MeSH
- aktivace enzymů fyziologie účinky léků MeSH
- financování organizované MeSH
- fosfolipasy A2, skupina VI metabolismus MeSH
- iontové kanály metabolismus MeSH
- krysa rodu rattus MeSH
- mastné kyseliny metabolismus MeSH
- mitochondriální ADP/ATP-translokasy metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie enzymologie účinky záření MeSH
- myokard enzymologie patologie MeSH
- oxidační stres fyziologie účinky léků MeSH
- oxidativní fosforylace MeSH
- potkani Wistar MeSH
- reaktivní formy kyslíku metabolismus MeSH
- reperfuzní poškození myokardu metabolismus patologie MeSH
- terc-butylhydroperoxid farmakologie MeSH
- viabilita buněk fyziologie MeSH
- zpětná vazba fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Cells with overactive RAS/protein kinase A (PKA) signaling, such as RAS2(Val19) cells, exhibit reduced proliferation rates and accelerated replicative senescence. We show here that the extended generation time of RAS2(Val19)cells is the result of abrogated ATP/ADP carrier activity of the mitochondria. Both PKA-dependent and independent routes are responsible for inhibiting ATP/ADP exchange in the RAS-overactive cells. The reduced carrier activity is due, at least in part, to elevated levels of reactive oxygen species (ROS), which also cause a proteolysis-dependent fragmentation of the Aac2p carrier both in vivo and on isolated mitochondria. Attenuated carrier activity is suppressed by overproducing the superoxide dismutase, Sod1p, and this enhances both the proliferation rate and the replicative longevity of RAS2(Val19) cells. In contrast, overproducing functional Aac2p restored proliferation but not longevity of RAS2(Val19) cells. Thus, Ras signaling affects proliferation rate and replicative lifespan by two different, ROS-dependent, routes. While the reduction in generation time is linked to the inactivation, specifically, of the mitochondrial nucleotide carrier, longevity is affected by other, and hitherto unknown, target(s) of ROS attack.
- MeSH
- aktivace enzymů MeSH
- down regulace MeSH
- financování organizované MeSH
- genetické inženýrství MeSH
- mitochondriální ADP/ATP-translokasy biosyntéza metabolismus nedostatek MeSH
- mutantní proteiny MeSH
- proliferace buněk MeSH
- proteinkinasy závislé na cyklickém AMP metabolismus MeSH
- ras proteiny genetika metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- Saccharomyces cerevisiae - proteiny biosyntéza MeSH
- Saccharomyces cerevisiae enzymologie genetika růst a vývoj MeSH
- signální transdukce MeSH
- stárnutí buněk MeSH
- superoxiddismutasa biosyntéza MeSH
- MeSH
- finanční podpora výzkumu jako téma MeSH
- krysa rodu rattus MeSH
- mitochondriální ADP/ATP-translokasy metabolismus MeSH
- mitochondrie fyziologie metabolismus MeSH
- receptory cytoplazmatické a nukleární genetika MeSH
- srdce fyziologie růst a vývoj MeSH
- transportní proteiny fyziologie genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
