Alena Vojtíšková

HIF and reactive oxygen species regulate oxidative phosphorylation in cancer.

A decrease in oxidative phosphorylation (OXPHOS) is characteristic of many cancer types and, in particular, of clear cell renal carcinoma (CCRC) deficient in von Hippel-Lindau (vhl) gene. In the absence of functional pVHL, hypoxia-inducible factor (HIF) 1-alpha and HIF2-alpha subunits are stabilized, which induces the transcription of many genes including those involved in glycolysis and reactive oxygen species (ROS) metabolism. Transfection of these cells with vhl is known to restore HIF-alpha subunit degradation and to reduce glycolytic genes transcription. We show that such transfection with vhl of 786-0 CCRC (which are devoid of HIF1-alpha) also increased the content of respiratory chain subunits. However, the levels of most transcripts encoding OXPHOS subunits were not modified. Inhibition of HIF2-alpha synthesis by RNA interference in pVHL-deficient 786-0 CCRC also restored respiratory chain subunit content and clearly demonstrated a key role of HIF in OXPHOS regulation. In agreement with these observations, stabilization of HIF-alpha subunit by CoCl(2) decreased respiratory chain subunit levels in CCRC cells expressing pVHL. In addition, HIF stimulated ROS production and mitochondrial manganese superoxide dismutase content. OXPHOS subunit content was also decreased by added H(2)O(2.) Interestingly, desferrioxamine (DFO) that also stabilized HIF did not decrease respiratory chain subunit level. While CoCl(2) significantly stimulates ROS production, DFO is known to prevent hydroxyl radical production by inhibiting Fenton reactions. This indicates that the HIF-induced decrease in OXPHOS is at least in part mediated by hydroxyl radical production.

Diminished synthesis of subunit a (ATP6) and altered function of ATP synthase and cytochrome c oxidase due to the mtDNA 2 bp microdeletion of TA at positions 9205 and 9206

Dysfunction of mitochondrial ATPase (F1F(o)-ATP synthase) due to missense mutations in ATP6 [mtDNA (mitochondrial DNA)-encoded subunit a] is a frequent cause of severe mitochondrial encephalomyopathies. We have investigated a rare mtDNA mutation, i.e. a 2 bp deletion of TA at positions 9205 and 9206 (9205DeltaTA), which affects the STOP codon of the ATP6 gene and the cleavage site between the RNAs for ATP6 and COX3 (cytochrome c oxidase 3). The mutation was present at increasing load in a three-generation family (in blood: 16%/82%/>98%). In the affected boy with severe encephalopathy, a homoplasmic mutation was present in blood, fibroblasts and muscle. The fibroblasts from the patient showed normal aurovertin-sensitive ATPase hydrolytic activity, a 70% decrease in ATP synthesis and an 85% decrease in COX activity. ADP-stimulated respiration and the ADP-induced decrease in the mitochondrial membrane potential at state 4 were decreased by 50%. The content of subunit a was decreased 10-fold compared with other ATPase subunits, and [35S]-methionine labelling showed a 9-fold decrease in subunit a biosynthesis. The content of COX subunits 1, 4 and 6c was decreased by 30-60%. Northern Blot and quantitative real-time reverse transcription-PCR analysis further demonstrated that the primary ATP6--COX3 transcript is cleaved to the ATP6 and COX3 mRNAs 2-3-fold less efficiently. Structural studies by Blue-Native and two-dimensional electrophoresis revealed an altered pattern of COX assembly and instability of the ATPase complex, which dissociated into subcomplexes. The results indicate that the 9205DeltaTA mutation prevents the synthesis of ATPase subunit a, and causes the formation of incomplete ATPase complexes that are capable of ATP hydrolysis but not ATP synthesis. The mutation also affects the biogenesis of COX, which is present in a decreased amount in cells from affected individuals.

Functional alteration of cytochrome c oxidase by SURF1 mutations in Leigh syndrome

Subacute necrotising encephalomyopathy (Leigh syndrome) due to cytochrome c oxidase (COX) deficiency is often caused by mutations in the SURF1 gene, encoding the Surf1 protein essential for COX assembly. We have investigated five patients with different SURF1 mutations resulting in the absence of Surf1 protein. All of them presented with severe and generalised COX defect. Immunoelectrophoretic analysis of cultured fibroblasts revealed 85% decrease of the normal-size COX complexes and significant accumulation of incomplete COX assemblies of 90-120 kDa. Spectrophotometric assay of COX activity showed a 70-90% decrease in lauryl maltoside (LM)-solubilised fibroblasts. In contrast, oxygen consumption analysis in whole cells revealed only a 13-31% decrease of COX activity, which was completely inhibited by detergent in patient cells but not in controls. In patient fibroblasts ADP-stimulated respiration was 50% decreased and cytofluorometry showed a significant decrease of mitochondrial membrane potential DeltaPsi(m) in state 4, as well as a 2.4-fold higher sensitivity of DeltaPsi(m) to uncoupler. We conclude that the absence of the Surf1 protein leads to the formation of incomplete COX complexes, which in situ maintain rather high electron-transport activity, while their H(+)-pumping is impaired. Enzyme inactivation by the detergent in patient cells indicates instability of incomplete COX assemblies.

Mitochondrial complex I inhibition in cerebral cortex of immature rats following homocysteic acid-induced seizures

The major finding of the present study concerns the marked decrease of respiratory chain complex I activity in the cerebral cortex of immature rats following seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side). This decrease was already evident during the acute phase of seizures (60-90 min after infusion) and persisted for at least 20 h after the seizures. It was selective for complex I since activities of complex II and IV and citrate synthase remained unaffected. Inhibition of complex I activity was not associated with changes in complex I content. Based on enhanced lipoperoxidation and decreased aconitase activity, it can be postulated that oxidative modification is most likely responsible for the observed inhibition. Mitochondrial respiration, as well as cortical ATP levels remained in the control range, apparently due to excess capacity of the complex I documented by energy thresholds. On the other hand, the enhanced production of reactive oxygen species by inhibited complex I was observed in mitochondria from HCA-treated animals. The decrease of complex I activity was substantially attenuated when animals were treated with substances providing an anticonvulsant effect and also with selected free radical scavengers. We can assume that inhibition of complex I may elicit enhanced formation of reactive oxygen species and contribute thus to neuronal injury demonstrated in this model.

Two components in pathogenic mechanism of mitochondrial ATPase deficiency: Energy deprivation and ROS production

Isolated defects of mitochondrial ATPase due to diminished biosynthesis of the enzyme represent new class of severe mitochondrial diseases of nuclear origin. The primary cause of decreased cellular content of ATPase appears to be a problem in assembly of the F1 catalytic part of the enzyme. With the aim to elucidate how the low ATPase content affects mitochondrial energy provision and ROS production, we have investigated fibroblasts from patients with ATPase decrease to 10-30%. Measurements of cellular respiration showed pronounced decrease in ATPase capacity for basal respiration, mitochondrial ATP synthesis was decreased to 26-33%. Cytofluorometric analysis using TMRM revealed altered discharge of mitochondrial membrane potential (DeltaPsim) in patient cells, which was 20 mV increased at state 3-ADP. Analysis of ROS production by CM-H2DCFDA demonstrated 2-fold increase in ROS production in patient cells compared to controls. ROS production rate was sensitive to uncoupler (FCCP) and thus apparently related to increased DeltaPsim. Our studies clearly demonstrate that low ATPase content and decreased mitochondrial ATP production lead to high values of DeltaPsim and are associated with activation of ROS generation by the mitochondrial respiratory chain. In conclusion, both the energetic deprivation and increased oxidative stress are important components of the pathogenic mechanism of ATPase disorders.

Mitochondrial Membrane Potential and ATP Production in Primary Disorders of ATP Synthase

Studies of fibroblasts with primary defects in mitochondrial ATP synthase (ATPase) due to heteroplasmic mtDNA mutations in the ATP6 gene, affecting protonophoric function or synthesis of subunit a, show that at high mutation loads, mitochondrial membrane potential ΔΨm at state 4 is normal, but ADP-induced discharge of ΔΨm is impaired and ATP synthesis at state 3-ADP is decreased. Increased ΔΨm and low ATP synthesis is also found when the ATPase content is diminished by altered biogenesis of the enzyme complex. Irrespective of the different pathogenic mechanisms, elevated ΔΨm in primary ATPase disorders could increase mitochondrial production of reactive oxygen species and decrease energy provision.