The role of the ATPase inhibitor factor 1 (IF1) in cancer cells adaptation to hypoxia and anoxia.

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF1, that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O2) unexpectedly showed that Δψm is preserved independently of the presence of IF1 and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing ΔµΗ+ with the FCCP uncoupler, the IF1-silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF1 overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF1 overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction.

Hypoxia decreases ROS level in human fibroblasts.

We have previously demonstrated that cells adapt to hypoxia using different metabolic reprogramming mechanisms depending on metabolism. We now investigate how the different adapting mechanisms affect reactive oxygen species (ROS) levels, and how ROS levels and cellular metabolism are linked. We show that when skin fibroblasts grew under short-term hypoxia (1% oxygen tension) ROS level markedly decreased (-50%) whatever substrate was available to the cells. Indeed, cellular ROS level linearly and directly decreased with oxygen tension. However, these relationships cannot explain the progressive ROS level decrease observed after prolonged cells hypoxia exposure. In glucose-enriched medium reduced mitochondrial mass and greater fragmentation are observed, both clear-cut indications of mitophagy suggesting that this is responsible for cellular ROS level decrease. Otherwise, in glucose-free medium exposure to prolonged hypoxia resulted in only minor mass reduction, but significantly enhanced expression of antioxidant enzymes. Interestingly, cellular ROS levels were lower in glucose-free compared to glucose-enriched medium under either normoxic or hypoxic conditions. Taken together, these findings reveal that in primary human fibroblasts hypoxia induces a decline in ROS and that different metabolism-dependent mechanisms contribute it, besides the major oxygen concentration decrease. In addition, the present data support the notion that metabolisms generating fewer ROS are associated with lower HIF-1α stabilization.

Is supercomplex organization of the respiratory chain required for optimal electron transfer activity?

The supra-molecular assembly of the main respiratory chain enzymatic complexes in the form of "super-complexes" has been proved by structural and functional experimental evidence. This evidence strongly contrasts the previously accepted Random Diffusion Model stating that the complexes are functionally connected by lateral diffusion of small redox molecules (i.e. Coenzyme Q and cytochrome c). This review critically examines the available evidence and provides an analysis of the functional consequences of the intermolecular association of the respiratory complexes pointing out the role of Coenzyme Q and of cytochrome c as channeled or as freely diffusing intermediates in the electron transfer activity of their partner enzymes.

Gradual alteration of mitochondrial structure and function by beta-amyloids: importance of membrane viscosity changes, energy deprivation, reactive oxygen species production, and cytochrome c release.

Intracellular amyloid beta-peptide (A beta) accumulation is considered to be a key pathogenic factor in sporadic Alzheimer's disease (AD), but the mechanisms by which it triggers neuronal dysfunction remain unclear. We hypothesized that gradual mitochondrial dysfunction could play a central role in both initiation and progression of sporadic AD. Thus, we analyzed changes in mitochondrial structure and function following direct exposure to increasing concentrations of A beta(1--42) and A beta(25--35) in order to look more closely at the relationships between mitochondrial membrane viscosity, ATP synthesis, ROS production, and cytochrome c release. Our results show the accumulation of monomeric A beta within rat brain and muscle mitochondria. Subsequently, we observed four different and additive modes of action of A beta, which were concentration dependent: (i) an increase in mitochondrial membrane viscosity with a concomitant decrease in ATP/O, (ii) respiratory chain complexes inhibition, (iii) a potentialization of ROS production, and (iv) cytochrome c release.

Increased state 4 mitochondrial respiration and swelling in early post-ischemic reperfusion of rat heart.

Isolated rat hearts were exposed to 30 min ischemia or to 30 min ischemia followed by 2, 5 or 40 min reperfusion and mitochondria were isolated at these different time points. ADP-stimulated, succinate-dependent respiration rate (state 3) was not significantly changed at the different time points examined. In contrast, state 4 (non-ADP-stimulated) respiration rate was significantly increased after 30 min ischemia, and it increased further during the first post-ischemic reperfusion period. Mitochondrial swelling, as evaluated under conditions of the major controlled ion channels (i.e. permeability transition pore and ATP-dependent mitochondrial K(+) channel) closed, significantly increased in parallel. It is suggested that the inner mitochondrial membrane permeability is increased under exposure of the heart to ischemia and early reperfusion, and that the phenomenon is reversible upon subsequent long periods of reperfusion.

Mechanisms for reduction of endothelial activation by oleate: inhibition of nuclear factor-kappaB through antioxidant effects.

In a model of early atherogenesis based on cultured endothelial cells, we observed that the incorporation of oleic acid in cellular lipids decreases the stimulated expression of several endothelial adhesion molecules and soluble products typically expressed during endothelial activation and involved in monocyte recruitment. We investigated possible mechanisms for this effect assessing the stimulated induction of nuclear factor-kappaB. In parallel, we also measured glutathione (GSH) content and the activity of antioxidant enzymes after oleate treatment and cytokine stimulation. Oleate prevented the stimulated depletion of GSH without any change in the activity of antioxidant enzymes. These results suggest an antioxidant mechanism by which oleate may exert direct vascular atheroprotective effects.

Dose-dependent inhibition of mitochondrial ATP synthase by 17 beta-estradiol.

Mitochondria produce energy through oxidative phosphorylation. A key enzyme in this pathway is F0F1-ATP synthase, catalyzing ATP production from ADP and inorganic phosphate. Recently a subunit of F0F1-ATP synthase, oligomycin sensitivity-conferring protein, was identified as a new estradiol-binding protein. Estradiol could directly modulate mitochondrial ATP synthase activity through this subunit. In addition, intracellular ATP levels play a role in apoptotic death, which is an energy-dependent process requiring functioning mitochondria. Here we examined the effect of 17 beta-estradiol on F0F1-ATP synthase directly (in permeabilized cells) and in intact osteoclastic FLG 29.1 cells, a model of inducible apoptosis. The baseline F0F1-ATP synthase activity of FLG 29.1 cells was 4.485 nmol/min per mg. Estradiol rapidly inhibited F0F1-ATP synthase activity in the physiological range (half-inhibition concentration, IC50, of 30 nmol/l). With 1 nmol/l of estradiol, the inhibition was already significant (8-10% inhibition, p < 0.01) and with 100 nmol/l residual enzyme activity was only 15% (85% inhibition, p < 0.01). In addition, the effect of estradiol appeared to be directed towards F0F1-ATP synthase, since succinate-sustained respiration, uncoupled from the electron transport chain, was unaffected by estradiol. We assayed F0F1-ATP synthase activity in FLG 29.1 cells during inducible apoptosis. No significant difference of ATP synthesis was detected in apoptotic cells versus controls. In conclusion, we showed a new non-genomic effect of estradiol on a key mitochondrial enzyme, which thereby directly modulates cellular energy metabolism.

Mitochondrial cytochrome c oxidase subunit III is selectively down-regulated by aluminum exposure in PC12S cells.

Aluminum (Al) has been implicated in several neurological diseases including dialysis dementia and Alzheimer's disease (AD). One possible mechanism of Al neurotoxicity could involve alteration of mitochondrial gene expression. We exposed PC12 cells to 0.1-100 microM AlCl3 for 6h at pH 7.4. Internalized Al, measured by atomic absorption spectrometry, was linearly proportional to the extracellular Al concentration. Northern blot analyses showed that cytochrome c oxidase subunit III (COX III) mRNA was significantly reduced by 70% after addition of 1 microM AlCl3. Higher concentrations of AlCl3 did not show a significant further effect. These results suggest that Al neurotoxicity involves a specific impairment of cytochrome c oxidase.

Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a.

We investigated the biochemical phenotype of the mtDNA T8993G point mutation in the ATPase 6 gene, associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families. All three carried >80% mutant genome in platelets and were manifesting clinically various degrees of the NARP phenotype. Coupled submitochondrial particles prepared from platelets capable of succinate-sustained ATP synthesis were studied using very sensitive and rapid luminometric and fluorescence methods. A sharp decrease (>95%) in the succinate-sustained ATP synthesis rate of the particles was found, but both the ATP hydrolysis rate and ATP-driven proton translocation (when the protons flow from the matrix to the cytosol) were minimally affected. The T8993G mutation changes the highly conserved residue Leu(156) to Arg in the ATPase 6 subunit (subunit a). This subunit, together with subunit c, is thought to cooperatively catalyze proton translocation and rotate, one with respect to the other, during the catalytic cycle of the F(1)F(0) complex. Our results suggest that the T8993G mutation induces a structural defect in human F(1)F(0)-ATPase that causes a severe impairment of ATP synthesis. This is possibly due to a defect in either the vectorial proton transport from the cytosol to the mitochondrial matrix or the coupling of proton flow through F(0) to ATP synthesis in F(1). Whatever mechanism is involved, this leads to impaired ATP synthesis. On the other hand, ATP hydrolysis that involves proton flow from the matrix to the cytosol is essentially unaffected.

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity.

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F1F0-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F1F0-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 +/- 3.0 sec in preconditioned vs. 83.4 +/- 4.4 sec in controls, p < 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.

The inhibition of endothelial activation by unsaturated fatty acids.

Dietary long-chain fatty acids (FA) may influence pathological processes involving endothelial activation and leukocyte-endothelial interactions, such as inflammation and atherosclerosis. We previously showed that the n-3 FA docosahexaenoate (22:6n-3, DHA) inhibits cytokine-stimulated expression of endothelial-leukocyte adhesion molecules and soluble cytokines in the range of nutritionally achievable plasma concentrations. More recently we assessed structural determinants of VCAM-1 inhibition by FA. Cultured endothelial cells were incubated first with various saturated, monounsaturated, n-6 or n-3 polyunsaturated FA alone and then together with interleukin-1 or tumor necrosis factor. Saturated FA did not inhibit cytokine-induced endothelial activation, while a progressive increase in inhibitory activity was observed, for the same chain length, with the increase in double bonds accompanying the transition from monounsaturates to n-6 and, further, to n-3 FA. Comparison of various FA indicated no role of the double-bond position or configuration; the greater number of double bonds could explain the greater inhibitory activity of n-3 vs. n-6 FA. In order to ascertain mechanisms for these effects, we demonstrated inhibition of nuclear factor-kappaB (NF-kappaB) activation by DHA in parallel with a reduction in hydrogen peroxide (a critical mediator of NF-kappaB activation) released by endothelial cells either extracellularly or intracellularly. This suggests that a property related to fatty acid peroxidability (the presence of multiple double bonds) is related to inhibitory properties of hydrogen peroxide release and, consequently, of endothelial activation.

Relevance of divalent cations to ATP-driven proton pumping in beef heart mitochondrial F0F1-ATPase.

The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F0F1-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me-ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the Km of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the Km value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F0F1-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the epsilon subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely.

Modification of the mitochondrial F1-ATPase epsilon subunit, enhancement of the ATPase activity of the IF1-F1 complex and IF1-binding dependence of the conformation of the epsilon subunit.

Treatment of bovine heart submitochondrial particles with a low concentration of 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent for the Trp residue of the epsilon subunit [Baracca, Barogi, Lenaz and Solaini (1993) Int. J. Biochem. 25, 1269-1275], enhances the ATP hydrolytic activity of the particles exclusively when the natural inhibitor protein IF1 is present. Similarly, isolated F1 [the catalytic sector of the mitochondrial H+-ATPase complex (ATP synthase)] treated with the reagent has the ATPase activity enhanced exclusively if IF1 is bound to it. These experiments suggest that the modification of the epsilon subunit decreases the inhibitory activity of IF1, eliciting the search for a relationship between the epsilon subunit and the inhibitory protein. Certainly, a reverse relationship exists because HNB binds covalently to the isolated F1 exclusively when the inhibitory protein is present. This finding is consistent with the existence of the epsilon subunit in different conformational states depending on whether IF1 is bound to F1 or not. Support for this assertion is obtained by measurements of the intrinsic phosphorescence decay rate of F1, a probe of the Trp epsilon subunit conformation in situ [Solaini, Baracca, Parenti-Castelli and Strambini (1993) Eur. J. Biochem. 214, 729-734]. A significant difference in phosphorescence decay rate is detected when IF1 is added to preparations of F1 previously devoid of the inhibitory protein. These studies indicate that IF1 and the epsilon subunit of the mitochondrial F1-ATPase complex are related, suggesting a possible role of the epsilon subunit in the mechanism of regulation of the mitochondrial ATP synthase.

Structural mapping of the epsilon-subunit of mitochondrial H(+)-ATPase complex (F1).

Phosphorescence and fluorescence energy transfer measurements have been used to locate the epsilon-subunit within the know structural frame of the mitochondrial soluble part of F-type H(+)-ATPase complex (F1). The fluorescence probe 2'-O-(trinitrophenyl)adenosine-5'-triphosphate was bound to the nucleotide binding sites of the enzyme, whereas the probe 7-diethylamino-3'-(4'-maleimidylphenyl)-4-methylcoumarin was attached to the single sulfhydryl residue of isolated oligomycin sensitivity-conferring protein (OSCP), which was then reconstituted with F1. Fluorescence and phosphorescence resonance energy transfer yields from the lone tryptophan residue of F1 present in the epsilon-polypeptide and the fluorescence labels attached to the F1 complex established that tryptophan is separated by 3.7 nm from Cys-118 of OSCP in the reconstituted OSCP-F1 complex, by 4.9 nm from its closest catalytic site and by more than 6.4 nm from the two other catalytic sites, including the lowest affinity ATP site. These separations together with the crystallographic coordinates of the F1 complex (Abrahams, J.P., A. G. W. Leslie, R. Lutter, and J.E. Walker. 1994. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370:621-628) place the epsilon-subunit in the stem region of the F1 molecule in a unique asymmetrical position relative to the catalytic sites of the enzyme.

Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing.

ATP hydrolase activity has been investigated in mitochondria from liver, heart, and skeletal muscle from adult (6 months) and aged (24 months) rats. No significant changes in total ATPase activity were observed in the three tissues, but the oligomycin sensitivity was slightly decreased in heart mitochondria of aged rats. The bicarbonate-induced stimulation of hydrolytic activity was somewhat decreased in mitochondria from aged rats, particularly in liver. No significant change was observed in ATPase activity after release of the endogenous inhibitor protein, IF1. It is concluded that no activity changes to be directly ascribed to the catalytic sector F1 of the enzyme occur upon ageing, but it cannot be excluded that changes in the membrane sector F0 occur as a consequence of mtDNA mutations.

Conformational changes of the mitochondrial F1-ATPase epsilon-subunit induced by nucleotide binding as observed by phosphorescence spectroscopy.

Changes in conformation of the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg2+ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the epsilon-subunit are evident at high ligand concentration added to both the nucleotide-depleted F1 (Nd-F1) and the F1 preparation containing the three tightly bound nucleotides (F1(2,1)). Since the effects observed are similar in both the F1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the epsilon-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F1. The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F1(3,0)). Phosphorescence lifetime measurements on this F1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the epsilon-subunit. This implies that the binding to the vacant noncatalytic site of F1(2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F1 and F1(2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F1(2,1) in the absence of Mg2+ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the epsilon-subunit is discussed.

A study of the mitochondrial F1-ATPase tryptophan phosphorescence at 273 K.

The bovine heart mitochondrial F1-ATPase complex exhibits an intrinsic tryptophan phosphorescence that can be used to monitor structural changes of the epsilon-subunit. The phosphorescence decay rate of F1 containing the tightly bound nucleotides increases upon addition of adenine nucleoside triphosphate in the presence of magnesium. The average phosphorescence lifetime of this enzyme preparation decreases from 10.2 to 7.8 ms upon Mg-ATP addition. Since increasing phosphorescence decay rate is related to increasing flexibility of proteins, Mg-ATP added to the F1-ATPase complex can enhance the flexibility of the protein structure surrounding the chromophore. Experiments carried out on F1 prepared with the three noncatalytic sites filled and the three catalytic sites vacant show a significant increase of the phosphorescence lifetime from 6.4 ms to 7.6 ms upon Mg-ATP addition. These results suggest that the mitochondrial F1-ATPase epsilon-subunit conformation senses differently the nucleoside triphosphate binding to catalytic or noncatalytic sites.

Dietary lipids and 5'-nucleotidase activity of rat cell plasma membranes.

Three groups of adult rats were fed black currant or olive oils or a 1:1 mixture of the two for three months. Feeding black currant oil diets, high in 18:2 (n-6), 18:3 (n-6), 18:3 (n-3), increased the heart and liver plasma membrane content of linoleic and arachidonic acid with a concomitant decrease of oleic acid. PUFA, n-3 and n-6 content and the bilayer lipid fluidity as examined by measuring the fluorescence anisotropy of diphenylhexatriene were not significantly affected. On the contrary, the 5'-nucleotidase of liver membrane of rats fed black currant diets was lower than that observed in membranes of liver from olive oil fed rats. Therefore it is concluded that PUFA and n-3/n-6 ratio as well as membrane fluidity do not influence the 5'-nucleotidase activity. It is suggested that the activity is sensitive to the amount of a specific fatty acid of the membrane (i.e., oleic or arachidonic acid) and/or to lipid supplementation which can influence the eicosanoids metabolism.

Interactions and effects of 2-hydroxy-5-nitrobenzyl bromide on the bovine heart mitochondrial F1-ATPase.

1. The F1-ATPase from bovine heart mitochondria was shown to chemically react and to absorb 2-hydroxy-5-nitrobenzyl bromide (HNB) with changes in catalytic properties. 2. The treatment of the enzyme with HNB at concentrations below 0.5 mM resulted in an increase of Vm and in an unchanged Km. Above 0.5 mM HNB elicited a concentration-dependent inhibition of F1. 3. HNB was found tightly bound to the enzyme epsilon-subunit whose tryptophan residue resulted modified. 4. The F1 activation appears the consequence of the covalent binding of the reagent to the enzyme, whilst inhibition results from non-covalent, reversible binding. 5. The possibility that the epsilon-subunit of mitochondrial F1-ATPase may influence the functional or regulating domain of the enzyme is discussed.

Tryptophan phosphorescence as a structural probe of mitochondrial F1-ATPase epsilon-subunit.

We report the detection of tryptophan phosphorescence emission from the sole residue in the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex. The phosphorescence spectrum, intensity and decay kinetics have been measured over the temperature range 160-273 K. The fine structure in the phosphorescence spectrum at low temperature, with the 0-0 vibrational band centered at 411 nm, reveals the hydrophobic nature of the chromophore's environment. Both the large width of the 0-0 vibrational band and the heterogeneous decay kinetics in fluid solution emphasize the existence of multiple conformations of the epsilon-subunit, structures which are rather stable as they do not interconvert in the millisecond time scale. Further, from the relatively long triplet lifetime at 273 K, it is possible to infer the existence of a tight, rigid core in the structure of the epsilon-subunit. Under subunit-dissociating conditions (6 M urea), the spectrum at 160 K undergoes a slight blue shift but since the phosphorescence lifetime, at all temperatures, is similar or longer than in the absence of dissociant, we conclude that dissociation does not lead to solvent exposure of the tryptophanyl side-chain. This conclusion is supported by the results obtained at 273 K by dissociating F1 in the presence of 0.3 M guanidine hydrochloride. Phosphorescence lifetimes indicate that 6 M urea leads to a more compact structure of the epsilon-subunit, whereas the opposite occurs when Mg-ATP is added to nucleotide-depleted F1. These spectroscopic changes establish unequivocally that the binding of the adenine nucleotide to the enzyme is accompanied by conformational changes involving the epsilon-subunit.