Dimerization interface and dynamic properties of yeast IF1 revealed by Site-Directed Spin Labeling EPR spectroscopy.

The mitochondrial ATPase inhibitor, IF1, regulates the activity of the mitochondrial ATP synthase. The oligomeric state of IF1 related to pH is crucial for its inhibitory activity. Although extensive structural studies have been performed to characterize the oligomeric states of bovine IF1, only little is known concerning those of yeast IF1. While bovine IF1 can be found as an inhibitory dimer at low pH and a non-inhibitory tetramer at high pH, a monomer/dimer equilibrium has been described for yeast IF1, high pH values favoring the monomeric state. Combining different strategies involving the grafting of nitroxide spin labels combined with Electron Paramagnetic Resonance (EPR) spectroscopy, the present study brings the first structural characterization, at the residue level, of yeast IF1 in its dimeric form. The results show that the dimerization interface involves the central region of the peptide revealing that the dimer corresponds to a non-inhibitory state. Moreover, we demonstrate that the C-terminal region of the peptide is highly dynamic and that this segment is probably folded back onto the central region. Finally, the pH-dependence of the inter-label distance distribution has been observed indicating a conformational change between two structural states in the dimer.

Proteomic analysis of F1F0-ATP synthase super-assembly in mitochondria of cardiomyoblasts undergoing differentiation to the cardiac lineage.

Mitochondria are essential organelles with multiple functions, especially in energy metabolism. An increasing number of data highlighted their role for cellular differentiation processes. We investigated differences in ATP synthase supra-molecular organization occurring in H9c2 cardiomyoblasts in the course of cardiac-like differentiation, along with ATP synthase biogenesis and maturation of mitochondrial cristae morphology. Using BN-PAGE analysis combined with one-step mild detergent extraction from mitochondria, a significant increase in dimer/monomer ratio was observed, indicating a distinct rise in the stability of the enzyme super-assembly. Remarkably, sub-stoichiometric mean values for ATP synthase subunit e were determined in both parental and cardiac-like H9c2 by an MS-based quantitative proteomics approach. This indicates a similar high proportion of complex molecules lacking subunit e in both cell types, and suggests a minor contribution of this component in the observed changes. 2D BN-PAGE/immunoblotting analysis and MS/MS analysis on single BN-PAGE band showed that the amount of inhibitor protein IF1 bound within the ATP synthase complexes increased in cardiac-like H9c2 and appeared greater in the dimer. In concomitance, a consistent improvement of enzyme activity, measured as both ATP synthesis and ATP hydrolysis rate, was observed, despite the increase of bound IF1 evocative of a greater inhibitory effect on the enzyme ATPase activity. The results suggest i) a role for IF1 in promoting dimer stabilization and super-assembly in H9c2 with physiological IF1 expression levels, likely unveiled by the fact that the contacts through accessory subunit e appear to be partially destabilized, ii) a link between dimer stabilization and enzyme activation.

Glucose-modulated mitochondria adaptation in tumor cells: a focus on ATP synthase and inhibitor Factor 1.

Warburg's hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking "positive" (activation/biogenesis) or "negative" (silencing) mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase ß subunit and Inhibitor Factor 1 (IF1). Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on ß-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

Mitochondrial bioenergetic profile and responses to metabolic inhibition in human hepatocarcinoma cell lines with distinct differentiation characteristics.

The classical view of tumour cell bioenergetics has been recently revised. Then, the definition of the mitochondrial profile is considered of fundamental importance for the development of anti-cancer therapies, but it still needs to be clarified. We investigated two human hepatocellular carcinoma cell lines: the partially differentiated HepG2 and the undifferentiated JHH-6. High resolution respirometry revealed a marked impairment/uncoupling of OXPHOS in JHH-6 compared with HepG2, with the phosphorylation system limiting the capacity for electron transport much more in JHH-6. Blocking glycolysis or mitochondrial ATP synthase we demonstrated that in JHH-6 ATP synthase functions in reverse and consumes glycolytic ATP, thereby sustaining ΔΨm. A higher expression level of ATP synthase Inhibitor Factor 1 (IF1), a higher extent of IF1 bound to ATP synthase and a lower ATPase/synthase capacity were documented in JHH-6. Thus, here IF1 appears to down-regulate the reverse mode of ATPsynthase activity, thereby playing a crucial role in controlling energy waste and ΔΨm. These results, while confirming the over-expression of IF1 in cancer cells, are the first to indicate an inverse link between cell differentiation status and IF1 (expression level and regulatory function).

Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) protects cells from oxidative stress via the base excision repair pathway and as a redox transcriptional coactivator. It is required for tumor progression/metastasis, and its up-regulation is associated with cancer resistance. Loss of APE1 expression causes cell growth arrest, mitochondrial impairment, apoptosis, and alterations of the intracellular redox state and cytoskeletal structure. A detailed knowledge of the molecular mechanisms regulating its different activities is required to understand the APE1 function associated with cancer development and for targeting this protein in cancer therapy. To dissect these activities, we performed reconstitution experiments by using wild-type and various APE1 mutants. Our results suggest that the redox function is responsible for cell proliferation through the involvement of Cys-65 in mediating APE1 localization within mitochondria. C65S behaves as a loss-of-function mutation by affecting the in vivo folding of the protein and by causing a reduced accumulation in the intermembrane space of mitochondria, where the import protein Mia40 specifically interacts with APE1. Treatment of cells with (E)-3-(2-[5,6-dimethoxy-3-methyl-1,4-benzoquinonyl])-2-nonyl propenoic acid, a specific inhibitor of APE1 redox function through increased Cys-65 oxidation, confirm that Cys-65 controls APE1 subcellular trafficking and provides the basis for a new role for this residue.

Cardiac differentiation promotes mitochondria development and ameliorates oxidative capacity in H9c2 cardiomyoblasts.

H9c2 undergoing cardiac differentiation induced by all-trans-retinoic acid were investigated for mitochondria structural features together with the implied functional changes, as a model for the study of mitochondrial development in cardiogenic progenitor cells. As the expression of cardiac markers became detectable, mitochondrial mass increased and mitochondrial morphology and ultrastructure changed. Reticular network organization developed and more bulky mitochondria with greater numbers of closely packed cristae and more electron-dense matrix were detected. Increased expression of PGC-1α proved the occurrence of mitochondrial biogenesis. Improvements in mitochondrial energetic competence were also documented, linked to better assembly between F(0) and F(1) sectors of the F(0)F(1)ATPsynthase enzyme complex.

The ectopic F(O)F(1) ATP synthase of rat liver is modulated in acute cholestasis by the inhibitor protein IF1.

Rat liver plasma membranes contain F(O)F(1) complexes (ecto-F(O)F(1)) displaying a similar molecular weight to the mitochondrial F(O)F(1) ATP synthase, as evidenced by Blue Native PAGE. Their ATPase activity was stably reduced in short-term extra-hepatic cholestasis. Immunoblotting and immunoprecipitation analyses demonstrated that the reduction in activity was not due to a decreased expression of ecto-F(O)F(1) complexes, but to an increased level of an inhibitory protein, ecto-IF(1), bound to ecto-F(O)F(1). Since cholestasis down regulates the hepatic uptake of HDL-cholesterol, and ecto-F(O)F(1) has been shown to mediate SR-BI-independent hepatic uptake of HDL-cholesterol, these findings provide support to the hypothesis that ecto-F(O)F(1) contributes to the fine control of reverse cholesterol transport, in parallel with SR-BI. No activity change of the mitochondrial F(O)F(1) ATP synthase (m-F(O)F(1)), or any variation of its association with m-IF(1) was observed in cholestasis, indicating that ecto-IF(1) expression level is modulated independently from that of ecto-F(O)F(1), m-IF(1) and m-F(O)F(1).

Cyclophilin D in mitochondrial pathophysiology.

Cyclophilins are a family of peptidyl-prolyl cis-trans isomerases whose enzymatic activity can be inhibited by cyclosporin A. Sixteen cyclophilins have been identified in humans, and cyclophilin D is a unique isoform that is imported into the mitochondrial matrix. Here we shall (i) review the best characterized functions of cyclophilin D in mitochondria, i.e. regulation of the permeability transition pore, an inner membrane channel that plays an important role in the execution of cell death; (ii) highlight new regulatory interactions that are emerging in the literature, including the modulation of the mitochondrial F1FO ATP synthase through an interaction with the lateral stalk of the enzyme complex; and (iii) discuss diseases where cyclophilin D plays a pathogenetic role that makes it a suitable target for pharmacologic intervention.

Cyclophilin D modulates mitochondrial F0F1-ATP synthase by interacting with the lateral stalk of the complex.

Blue native gel electrophoresis purification and immunoprecipitation of F(0)F(1)-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F(1) sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.

Mitochondrial and cell-surface F0F1ATPsynthase in innate and acquired cardioprotection.

Mitochondria are central to heart function and dysfunction, and the pathways activated by different cardioprotective interventions mostly converge on mitochondria. In a context of perspectives in innate and acquired cardioprotection, we review some recent advances in F(0)F(1)ATPsynthase structure/function and regulation in cardiac cells. We focus on three topics regarding the mitochondrial F(0)F(1)ATPsynthase and the plasma membrane enzyme, i.e.: i) the crucial role of cardiac mitochondrial F(0)F(1)ATPsynthase regulation by the inhibitory protein IF(1) in heart preconditioning strategies; ii) the structure and function of mitochondrial F(0)F(1)ATPsynthase oligomers in mammalian myocardium as possible endogenous factors of mitochondria resistance to ischemic insult; iii) the external location and characterization of plasma membrane F(0)F(1) ATP synthase in search for possible actors of its regulation, such as IF(1) and calmodulin, at cell surface.

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F(0)F(1)ATP synthase.

The role of the integral inner membrane subunit e in self-association of F(0)F(1)ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F(0)F(1)ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, gamma and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits gamma and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F(0)F(1)ATP synthase.

Mammalian ATPsynthase monomer versus dimer profiled by blue native PAGE and activity stain.

Studies into the effects of oligomerization on F(0)F(1)ATPsynthase function are contradictory. We optimized the in-gel ATPase assay to investigate the functional differences of monomers versus dimers. In Triton X-100 extracts of heavy bovine heart mitochondria (HBHM) and mitoplasts, but not submitochondrial particles (MgATP-SMP), dimers had greater specific activity than monomers: at 30 degrees C, the dimer/monomer activity ratios were 2.3, 1.4, and 1.0, respectively. These differences in HBHM and mitoplasts extracts were enhanced at 37 degrees C but lost at 20 degrees C. In mitoplasts but not in MgATP-SMP, dimers were selectively shielded from limited chymotrypsin degradation of F(1) alpha subunit, possibly due to interactions with other proteins or ligands in the native inner membrane. Despite these differences, all three preparations had similar percentages of dimers and similar contents of the native inhibitor IF(1) in Vm (monomer) and (dimer) Vd. These results suggest that, in native membrane, monomers and dimers are functionally distinct.

Characterization of oligomeric forms from mammalian F0F1ATP synthase by BN-PAGE: the role of detergents.

It is now widely accepted that F0F1ATPsynthase is present in membrane, beside as monomers, in homo-dimeric and higher homo-oligomeric forms, which probably play critical roles in determining mitochondrial morphology. One-step mild detergent extraction followed by blue native electrophoresis (BN-PAGE) is a very interesting tool for studying the native membrane protein assemblies which can be associated with second/third-dimensional SDS-PAGE, immunoblotting, in-gel enzyme activity staining and mass spectrometry analyses. By combining these techniques, we resolved monomers and higher oligomeric forms of ATPsynthase from bovine heart mitochondria. However, a critical point is the choice of the detergents, which strongly influence the protein pattern of BN-PAGE. By using Triton X-100 we obtained that, in spite of the same subunit composition, monomers have a much lower specific activity than dimers and the two forms have a different pattern of tyrosine phosphorylation, suggesting that monomers and dimers are functionally distinct in membrane. In addition, enzyme self-association appeared to occur independently from the binding to ATPsynthase of the inhibitor protein IF1. Dodecylmaltoside was optimal to extract the enzyme from single biopsy samples, allowing us to demonstrate that IF1 plays a central role in regulating the enzyme activity in heart in vivo. Only low concentration of digitonin maintained significant amounts of ATPsynthase oligomers, which seemed to retain intact their native catalytic properties.

Differential steady-state tyrosine phosphorylation of two oligomeric forms of mitochondrial F0F1ATPsynthase: a structural proteomic analysis.

We investigated tyrosine phosphorylation of F(0)F(1)ATPsynthase using 3-D blue native (BN)-SDS-PAGE, a refinement of the electrophoretic analysis of mitochondrial complexes. Bovine heart mitochondria were detergent-solubilized and subjected to BN-PAGE. Bands of ATPsynthase monomer (Vmon) and dimer (Vdim) were excised and submitted to SDS-PAGE and immunoblotting. One protein corresponding to F(1)gamma subunit was detected by anti-phosphotyrosine antibody in monomer but not in dimer. This was confirmed by MS peptide mapping. LC-ESI/MS analysis after 3-D SDS-PAGE demonstrated phosphotyrosine in fragment 43-54. NetPhos scores predicted the phosphorylated residue to be Tyr52, in a solvent-accessible loop at the foot of the F(1) central stalk.

Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber's hereditary optic neuropathy.

Oxidative stress and imbalance between free radical generation and detoxification may play a pivotal role in the pathogenesis of Leber's hereditary optic neuropathy (LHON). Mitochondria, carrying the homoplasmic 11778/ND4, 3460/ND1 and 14484/ND6 mtDNA point mutations associated with LHON, were used to generate osteosarcoma-derived cybrids. Enhanced mitochondrial production of reactive oxygen species has recently been demonstrated in these cybrids [Beretta S, Mattavelli L, Sala G, Tremolizzo L, Schapira AHV, Martinuzzi A, Carelli V & Ferrarese C (2004) Brain 127, 2183-2192]. The aim of this study was to characterize the antioxidant defences of these LHON-affected cells. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutases (SOD) and catalase, and the amounts of glutathione (GSH) and oxidized glutathione (GSSG) were measured in cybrids cultured both in glucose-rich medium and galactose-rich medium. The latter is known to cause oxidative stress and to trigger apoptotic death in these cells. In spite of reduced SOD activities in all LHON cybrids, and of low GPx and GR activities in cells with the most severe 3460/ND1 and 11778/ND4 mutations, GSH and GSSG content were not significantly modified in LHON cybrids cultured in glucose medium. In contrast, in galactose, GSSG concentrations increased significantly in all cells, indicating severe oxidative stress, whereas GR and MnSOD activities further decreased in all LHON cybrids. These data suggest that, in cells carrying LHON mutations, there is a decrease in antioxidant defences, which is especially evident in cells with mutations associated with the most severe clinical phenotype. This is magnified by stressful conditions such as exposure to galactose.