Mitochondrial Adenine Nucleotide Research Articles

Assessment of Membrane-Bound Mammal Mitochondrial Adenine Nucleotide Translocase Topography by Experimental Antibodies

To gain insight into the immunogenicity of mitochondrial adenine nucleotide translocase (ANT), we raised antibodies against purified bovine heart ANT by induction of ascitic fluid in male Balb/c mice. We identified the antigenic determinants detected by these antibodies by (1) immunodetection of GST-ANT fusion proteins and selected partial constructs of ANT, (2) immunodetection of chemically synthesized overlapping peptides on solid support, and (3) back-titration ELISA. Results revealed a short epitope spreading of the antibodies, resulting in a small number of antigenic determinants. Thus, each antibody detects one or two major epitopes located in the putative hydrophilic loops M2 and M3. No evidence for the antigenicity of the first 133 amino acids of ANT was obtained. These well-characterized antibodies were used to study the topography of the membrane-bound ANT by back-titration ELISA with mitochondrial membranes. We demonstrated that amino acids 145-150 and 230-237 are fully accessible to the antibodies in native ANT, whereas regions 133-140 and 244-251 are not. Furthermore, we used mitochondria devoid of the outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) to establish the matrix or cytosolic orientation of loops M2 and M3. The results clearly show that these loops have a matrix orientation and thus support the six transmembrane segment model of ANT topography in the inner mitochondrial membrane.

The human mitochondrial ADP/ATP carriers: kinetic properties and biogenesis of wild-type and mutant proteins in the yeast S. cerevisiae.

The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.

Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria

The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.

Adenine nucleotide translocator 1 deficiency associated with Sengers syndrome.

Sengers syndrome is characterized by congenital cataracts, hypertrophic cardiomyopathy, mitochondrial myopathy, and lactic acidosis, but no abnormalities have been found with routine mitochondrial biochemical diagnostics (the determination of pyruvate oxidation rates and enzyme measurements). In immunoblot analysis, the protein content of the mitochondrial adenine nucleotide translocator 1 (ANT1) was found to be strongly reduced in the muscle tissues of two unrelated patients with Sengers syndrome. In addition, low residual adenine nucleotide translocator activity was detected upon the reconstitution of detergent-solubilized mitochondrial extracts from the patients' skeletal or heart muscle into liposomes. Sequence analysis and linkage analysis showed that ANT1 was not the primary genetic cause of Sengers syndrome. We propose that transcriptional, translational, or posttranslational events are responsible for the ANT1 deficiency associated with the syndrome.

Epitope Mapping of Mitochondrial Adenine Nucleotide Translocase-1 in Idiopathic Dilated Cardiomyopathy

Mitochondrial adenine nucleotide translocase (ANT) is a specific target for the autoantibody response in idiopathic dilated cardiomyopathy (IDCM). We have undertaken an epitope analysis of ANT in IDCM by immunoblot with recombinant GST-ANT fusion proteins and with cellulose-bound decapeptides of human ANT1. Forty-five patients with IDCM, 17 patients with ischemic left ventricle dysfunction (LVD) and 20 controls were analyzed for circulating antibodies against ANT (AAb-ANT). Sixteen of the 45 (36%) IDCM patients showed AAb-ANT above controls. In immunoblots, AAb-ANT detected purified bovine heart ANT and GST-ANT1 and GST-ANT2 isoforms and, less frequently, the GST-ANT3 isoform. A construct lacking the last 146 amino acids did not react with AAb-ANT, indicating that the main epitopes are in the C-terminal 146 amino acids. Immunodetection of decapeptides covering this region shows that AAb-ANT detects at least three epitopes, demonstrating that ANT is the primary target of AAb-ANT. The most significant epitopes belong to the M2 and M3 hydrophilic loops of ANT suggesting that apart from being essential for its activity, these loops are highly immunogenic.

A custom microarray analysis of gene expression during programmed cell death in Arabidopsis thaliana.

Programmed cell death (PCD) is a form of cellular suicide requiring active gene expression, and occurs in both animals and plants. While the cascade of events and the genes that control PCD have been extensively studied in animals, we remain largely ignorant about the similar process in plant cells. Many of the key proteins of animal cell death such as the Bcl-2 family and the caspase family of proteases do not appear to be conserved in plants, suggesting that plants may employ unique mechanisms to execute PCD. To identify genetic elements of PCD in plants, we monitored changes in transcript levels of approximately 100 selected genes during cell death in an Arabidopsis cell suspension culture using a cDNA microarray. PCD was induced in the cell cultures by two independent means (heat treatment or by allowing the cultures to senesce) to allow the distinction to be drawn between changes in gene expression that are related to PCD and those that are specific to a particular treatment. We argue that genes whose expression is altered during PCD induced by two different means may be generally involved in all types of PCD. We show that certain oxidative stress-related genes, including CSD1, CSD3, and GPX, in addition to cysteine proteinases, some transcription factors, and HR-related genes may serve as markers of a core plant cell death programme. Additionally we observe a down-regulation of the mitochondrial adenine nucleotide transporter and suggest that this may be an early event in the execution of plant PCD.

Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria.

The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.

Diazoxide and pinacidil uncouple pyruvate-malate-induced mitochondrial respiration.

We investigated the effects of K(ATP) channel openers diazoxide and pinacidil on the respiration rate and membrane potential (deltapsi) of rat heart mitochondria, oxidizing pyruvate and malate. Diazoxide and pinacidil (58.8-1348.3 microM) increased the V2 (-ADP) respiration rate accordingly by 13-208% and 30-273% and decreased the deltapsi by 2-17% and 6-55%. These effects were also similar in the respiration medium without K+. Moreover, carboxyatractyloside completely abolished diazoxide- and pinacidil-induced uncoupling, indicating a role for the mitochondrial adenine nucleotide translocase in this process.

Defects of intergenomic communication: autosomal disorders that cause multiple deletions and depletion of mitochondrial DNA

Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a growing number of autosomal diseases that have been classified as defects of intergenomic communication. MNGIE, an autosomal recessive disorder associated with mtDNA alterations is due to mutations in thymidine phosphorylase that may cause imbalance of the mitochondrial nucleotide pool. Subsequently, mutations in the mitochondrial proteins adenine nucleotide translocator 1, Twinkle, and polymerase gamma have been found to cause autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA. Uncovering the molecular bases of intergenomic communication defects will enhance our understanding of the mechanisms responsible for maintaining mtDNA integrity.

Mitochondrial Creatine Kinase and Mitochondrial Outer Membrane Porin Show a Direct Interaction That Is Modulated by Calcium

Mitochondrial creatine kinase (MtCK) co-localizes with mitochondrial porin (voltage-dependent anion channel) and adenine nucleotide translocator in mitochondrial contact sites. A specific, direct protein-protein interaction between MtCK and mitochondrial porin was demonstrated using surface plasmon resonance spectroscopy. This interaction was independent of the immobilized binding partner (porin reconstituted in liposomes or MtCK) or the analyzed isoform (chicken sarcomeric MtCK or human ubiquitous MtCK, human recombinant porin, or purified bovine porin). Increased ionic strength reduced the binding of MtCK to porin, suggesting predominantly ionic interactions. By contrast, micromolar concentrations of Ca(2+) increased the amount of bound MtCK, indicating a physiological regulation of complex formation. No interaction of MtCK with reconstituted adenine nucleotide translocator was detectable in our experimental setup. The relevance of these findings for structure and function of mitochondrial contact sites is discussed.

Cardioprotection by Ischemic Preconditioning Preserves Mitochondrial Function and Functional Coupling Between Adenine Nucleotide Translocase and Creatine Kinase

M. N. Laclau, S. Boudina, J. B. Thambo, L. Tariosse, G. Gouverneur, S. Bonoron-Adèle, V. A. Saks, K. D. Garlid and P. Dos Santos. Cardioprotection by Ischemic Preconditioning Preserves Mitochondrial Function and Functional Coupling Between Adenine Nucleotide Translocase and Creatine Kinase. Journal of Molecular and Cellular Cardiology (2001)33 , 947–956. This study investigates the effect of ischemic preconditioning on mitochondrial function, including functional coupling between the adenine nucleotide translocase and mitochondrial creatine kinase, which is among the first reactions to be altered in ischemia. Three groups of Langendorff-perfused rat hearts were studied: a control group, a group subjected to 30 min ischemia followed by 15 min reperfusion, and a group subjected to ischemic preconditioning prior to 30 min ischemia and 15 min reperfusion. Ischemic preconditioning significantly delayed the onset and amplitude of contracture during ischemia, decreased enzymatic release, and improved the recovery of heart contractile function after reperfusion. Mitochondrial function was assessed in permeabilized skinned fibers. The protective effect of preconditioning was associated with preservation of mitochondrial function, as evidenced by maintenance of the high K1/2for ADP in regulation of mitochondrial respiration and Vmaxof respiration, the near absence of respiratory stimulation by exogenous cytochrome c, and preservation of functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase. These data suggest that ischemic preconditioning preserves the structure–function of the intermembrane space, perhaps by opening the mitochondrial ATP-sensitive K+channel. The consequence is preservation of energy transfer processes from mitochondria to ATP-utilizing sites in the cytosol. Both of these factors may contribute to cardioprotection and better functional recovery of preconditioned hearts.

Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells

Hepatic stellate cell (HSC) transformation and proliferation play an important role in liver fibrogenesis, and HSC apoptosis may be involved in the termination of this response. Expression of the peripheral benzodiazepine receptor (PBR) and PBR-ligand-induced apoptosis were studied in cultured rat liver HSC. Transformation of HSC led to a transient expression of PBR at the messenger RNA and protein level, which was maximal after about 3 and 7 days of culture, respectively, and declined thereafter. Immunoreactive PBR showed a punctate staining and colocalized with mitochondrial manganese-dependent superoxide dismutase and adenine nucleotide translocator 1. The selective PBR ligands 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and 4' chlorodiazepam (Ro5-4864), but not the centrally acting benzodiazepine ligand clonazepam, induced dose-dependent apoptosis in HSC. The apoptotic potency of PK11195 paralleled the level of PBR expression. PK11195 induced dephosphorylation of protein kinase B/Akt and Bad and a downregulation of Bcl-2. Collapse of the mitochondrial membrane potential preceeded PBR-ligand-induced apoptosis. No apoptosis was induced by PK11195 in parenchymal cells, despite the presence of PBR, and PK11195 had no effect in these cells on Bad phosphorylation and Bcl-2 expression. Transformation of HSC leads to a transient expression of PBR and renders the cells sensitive to PBR-ligand-induced apoptosis, involving protein kinase B/Akt and Bad-dependent mechanisms.

Blocking ICOS in combination with CD28 impairs the proper activation of aggressor Th1 cells but is not necessary for suppression by regulatory T cells

<h2>Abstract</h2> <b>Background & Aims:</b> Hepatic stellate cell (HSC) transformation and proliferation play an important role in liver fibrogenesis, and HSC apoptosis may be involved in the termination of this response. <b>Methods:</b> Expression of the peripheral benzodiazepine receptor (PBR) and PBR-ligand–induced apoptosis were studied in cultured rat liver HSC. <b>Results:</b> Transformation of HSC led to a transient expression of PBR at the messenger RNA and protein level, which was maximal after about 3 and 7 days of culture, respectively, and declined thereafter. Immunoreactive PBR showed a punctate staining and colocalized with mitochondrial manganese-dependent superoxide dismutase and adenine nucleotide translocator 1. The selective PBR ligands 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and 4' chlorodiazepam (Ro5-4864), but not the centrally acting benzodiazepine ligand clonazepam, induced dose-dependent apoptosis in HSC. The apoptotic potency of PK11195 paralleled the level of PBR expression. PK11195 induced dephosphorylation of protein kinase B/Akt and Bad and a downregulation of Bcl-2. Collapse of the mitochondrial membrane potential preceeded PBR-ligand–induced apoptosis. No apoptosis was induced by PK11195 in parenchymal cells, despite the presence of PBR, and PK11195 had no effect in these cells on Bad phosphorylation and Bcl-2 expression. <b>Conclusions:</b> Transformation of HSC leads to a transient expression of PBR and renders the cells sensitive to PBR-ligand–induced apoptosis, involving protein kinase B/Akt and Bad-dependent mechanisms. GASTROENTEROLOGY 2001;120:1212-1226

Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death.

Activation of terminal caspases such as caspase-3 plays an important role in the execution of neuronal cell death after transient cerebral ischemia. Although the precise mechanism by which terminal caspases are activated in ischemic neurons remains elusive, recent studies have postulated that the mitochondrial cell death-signaling pathway may participate in this process. The bcl-2 family member protein Bax is a potent proapoptotic molecule that, on translocation from cytosol to mitochondria, triggers the activation of terminal caspases by increasing mitochondrial membrane permeability and resulting in the release of apoptosis-promoting factors, including cytochrome c. In the present study, the role of intracellular Bax translocation in ischemic brain injury was investigated in a rat model of transient focal ischemia (30 minutes) and reperfusion (1 to 72 hours). Immunochemical studies revealed that transient ischemia induced a rapid translocation of Bax from cytosol to mitochondria in caudate neurons, with a temporal profile and regional distribution coinciding with the mitochondrial release of cytochrome c and caspase-9. Further, in postischemic caudate putamen in vivo and in isolated brain mitochondria in vitro, the authors found enhanced heterodimerization between Bax and the mitochondrial membrane permeabilization-related proteins adenine nucleotide translocator (ANT) and voltage-dependent anion channel. The ANT inhibitor bongkrekic acid prevented Bax and ANT interactions and inhibited Bax-triggered caspase-9 release from isolated brain mitochondria in vitro. Bongkrekic acid also offered significant neuroprotection against ischemia-induced caspase-3 and caspase-9 activation and cell death in the brain. These results strongly suggest that the Bax-mediated mitochondrial apoptotic signaling pathway may play an important role in ischemic neuronal injury.

The physiological role of the creatine kinase system: evolution of views.

The development of ideas concerning the buffer and transport functions of the creatine kinase system is described. The concept of ATP compartmentation at sites of its production and utilization is critically analyzed. Kinetic, thermodynamic, and structural data used as a basis for a hypothesis on the structural and functional coupling of mitochondrial creatine kinase and adenine nucleotide translocase are comprehensively analyzed, and experimental evidence inconsistent with this hypothesis is presented. It seems that the mitochondrial creatine kinase may serve to equilibrate ADP concentration in the intermembrane space with fluctuating ADP concentrations in the cytoplasm. It is suggested that creatine kinase molecules bound to other intracellular structures (e.g., to myofibrils) may equilibrate local ADP concentrations with those present in the cytoplasm.

Distinct Effects of Saturated and Monounsaturated Fatty Acids on β-Cell Turnover and Function

Glucotoxicity and lipotoxicity contribute to the impaired beta-cell function observed in type 2 diabetes. Here we examine the effect of saturated and unsaturated fatty acids at different glucose concentrations on beta-cell proliferation and apoptosis. Adult rat pancreatic islets were cultured onto plates coated with extracellular matrix derived from bovine corneal endothelial cells. Exposure of islets to saturated fatty acid (0.5 mmol/l palmitic acid) in medium containing 5.5, 11.1, or 33.3 mmol/l glucose for 4 days resulted in a five- to ninefold increase of beta-cell DNA fragmentation. In contrast, monounsaturated palmitoleic acid alone (0.5 mmol/l) or in combination with palmitic acid (0.25 or 0.5 mmol/l each) did not affect DNA fragmentation. Increasing concentrations of glucose promoted beta-cell proliferation that was dramatically reduced by palmitic acid. Palmitoleic acid enhanced the proliferation activity in medium containing 5.5 mmol/l glucose but had no additional effect at higher glucose concentrations (11.1 and 33.3 mmol/l). The cell-permeable ceramide analog C2-ceramide mimicked both the palmitic acid-induced beta-cell apoptosis and decrease in proliferation. Moreover, the ceramide synthetase inhibitor fumonisin B1 blocked the deleterious effects of palmitic acid on beta-cell viability. Additionally, palmitic acid but not palmitoleic acid decreased the expression of the mitochondrial adenine nucleotide translocator and induced release of cytochrome c from the mitochondria into the cytosol. Finally, palmitoleic acid improved beta-cell-secretory function that was reduced by palmitic acid. Taken together, these results suggest that the lipotoxic effect of the saturated palmitic acid involves an increased apoptosis rate coupled with reduced proliferation capacity of beta-cells and impaired insulin secretion. The deleterious effect of palmitate on beta-cell turnover is mediated via formation of ceramide and activation of the apoptotic mitochondrial pathway. In contrast, the monounsaturated palmitoleic acid does not affect beta-cell apoptosis, yet it promotes beta-cell proliferation at low glucose concentrations, counteracting the negative effects of palmitic acid as well as improving beta-cell function.

γ-Glutamyltranspeptidase–Deficient Knockout Mice as a Model to Study the Relationship Between Glutathione Status, Mitochondrial Function, and Cellular Function

γ-Glutamyltranspeptidase (GGT)-deficient mice (GGT−/−) display chronic glutathione (GSH) deficiency, growth retardation, and die at a young age (<20 weeks). Using livers from these mice, we investigated the relationship between GSH content, especially mitochondrial, and mitochondrial and cellular function. We found that the GSH content of isolated liver mitochondria was diminished by ≥50% in GGT−/− mice when compared with wild-type mice. Respiratory control ratios (RCRs) of GGT−/− mice liver mitochondria were ≤60% those of wild-type mice primarily as a result of impaired state 3 respiration. Mitochondrial adenine nucleotide content was decreased by ≥40% in mitochondria obtained from GGT−/− mice. We observed a strong correlation between mitochondrial GSH content and RCRs. Even moderate decreases (<50%) correlated with adverse effects with respect to respiration. Electron microscopy revealed that livers from GGT−/− knockout mice were deprived of fat and glycogen, and swollen mitochondria were observed in animals that were severely deprived of GSH. Thus, GGT−/− mice exhibit a loss of GSH homeostasis and impaired oxidative phosphorylation, which may be related to the rate of adenosine triphosphate (ATP) formation and subsequently leads to progressive liver injury, which characterizes the diseased state. We also found that supplementation of GGT−/− mice with N-acetylcysteine (NAC) partially restored liver GSH, but fully restored mitochondrial GSH and respiratory function. Electron microscopy revealed that the livers of NAC-supplemented GGT−/− mice contained fat and glycogen; however, slightly enlarged mitochondria were found in some livers. NAC supplementation did not have any beneficial effect on the parameters examined in wild-type mice.(Hepatology 2000;32:740-749.)

Thyroid Hormone Coordinates Respiratory Control Maturation and Adenine Nucleotide Translocator Expression in Heart In Vivo

The signal transduction mechanism linking mitochondrial ATP synthesis with cytosolic ATP utilization in heart changes during postnatal development in vivo. This maturational process occurs in parallel with accumulation of mitochondrial adenine nucleotide translocator (ANT), which provides a possible site for respiratory control. We postulated that thyroid hormone regulates these maturational processes. We used (31)P MR spectroscopy to determine the relationship between myocardial high-energy phosphates, phosphocreatine, and ADP and oxygen consumption (MVO(2)) during epinephrine stimulation in 32- to 40-day-old lambs thyroidectomized after birth (THY) and age-matched controls. Steady-state protein and mRNA levels for ANT isoforms and beta-F(1)-ATPase were assessed from left ventricular tissues by Western and Northern blotting. With greater doses of epinephrine, THY attained lower peak MVO(2) than controls (P:<0.05). Controls maintained high-energy phosphate levels, unlike THY, which demonstrated significantly decreased phosphocreatine/ATP and increased cytosolic ADP despite lower peak MVO(2). No significant differences in beta-F(1)-ATPase protein or mRNA occurred between groups. However, ANT isoform mRNA levels were 2-fold greater and protein levels 4-fold greater in control hearts. These data imply that the maturational shift away from ADP-mediated respiratory control is regulated by thyroid hormone in vivo. Specific thyroid-modulated increases in ANT mRNA and protein imply that this regulation occurs in part at a pretranslational level.

Role of oxidative phosphorylation in Bax toxicity.

The Bcl-2-related protein Bax is toxic when expressed either in yeast or in mammalian cells. Although the mechanism of this toxicity is unknown, it appears to be similar in both cell types and dependent on the localization of Bax to the outer mitochondrial membrane. To investigate the role of mitochondrial respiration in Bax-mediated toxicity, a series of yeast mutant strains was created, each carrying a disruption in either a component of the mitochondrial electron transport chain, a component of the mitochondrial ATP synthesis machinery, or a protein involved in mitochondrial adenine nucleotide exchange. Bax toxicity was reduced in strains lacking the ability to perform oxidative phosphorylation. In contrast, a respiratory-competent strain that lacked the outer mitochondrial membrane Por1 protein showed increased sensitivity to Bax expression. Deficiencies in other mitochondrial proteins did not affect Bax toxicity as long as the ability to perform oxidative phosphorylation was maintained. Characterization of Bax-induced toxicity in wild-type yeast demonstrated a growth inhibition that preceded cell death. This growth inhibition was associated with a decreased ability to carry out oxidative phosphorylation following Bax induction. Furthermore, cells recovered following Bax-induced growth arrest were enriched for a petite phenotype and were no longer able to grow on a nonfermentable carbon source. These results suggest that Bax expression leads to an impairment of mitochondrial respiration, inducing toxicity in cells dependent on oxidative phosphorylation for survival. Furthermore, Bax toxicity is enhanced in yeast deficient in the ability to exchange metabolites across the outer mitochondrial membrane.

Interaction among mitochondria, mitogen-activated protein kinases, and nuclear factor-kappaB in cellular models of Parkinson's disease.

Oxidative stress induced by acute complex I inhibition with 1-methyl-4-phenylpyridinium ion activated biphasically the stress-activated c-Jun N-terminal kinase (JNK) and the early transcription factor nuclear factor-kappaB (NF-kappaB) in SH-SY5Y neuroblastoma cells. Early JNK activation was dependent on mitochondrial adenine nucleotide translocator (ANT) activity, whereas late-phase JNK activation and the cleavage of signaling proteins Raf-1 and mitogen-activated protein kinase (MAPK) kinase (MEK) kinase (MEKK)-1 appeared to be ANT-independent. Early NF-kappaB activation depended on MEK, later activation required an intact electron transport chain (ETC), and Parkinson's disease (PD) cybrid (mitochondrial transgenic cytoplasmic hybrid) cells had increased basal NF-kappaB activation. Mitochondria appear capable of signaling ETC impairment through MAPK modules and inducing protective NF-kappaB responses, which are increased by PD mitochondrial genes amplified in cybrid cells. Irreversible commitment to apoptosis in this cell model may derive from loss of Raf-1 and cleavage/activation of MEKK-1, processes reported in other models to be caspase-mediated. Therapeutic strategies that reduce mitochondrial activation of proapoptotic MAPK modules, i.e., JNK, and enhance survival pathways, i.e., NF-kappaB, may offer neuroprotection in this debilitating disease.