[Show abstract][Hide abstract] ABSTRACT: Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting as early-onset mitochondrial encephalo-cardiomyopathies. Up to now, mutations in four nuclear genes were associated with isolated deficiency of ATP synthase. Two of them, ATP5A1 and ATP5E encode enzyme's structural subunits alpha and epsilon, respectively, while the other two ATPAF2 and TMEM70 encode specific ancillary factors that facilitate the biogenesis of ATP synthase. All these defects share a similar biochemical phenotype with pronounced decrease in the content of fully assembled and functional ATP synthase complex. However, substantial differences can be found in their frequency, molecular mechanism of pathogenesis, clinical manifestation as well as the course of the disease progression. While for TMEM70 the number of reported patients as well as spectrum of the mutations is steadily increasing, mutations in ATP5A1, ATP5E and ATPAF2 genes are very rare. Apparently, TMEM70 gene is highly prone to mutagenesis and this type of a rare mitochondrial disease has a rather frequent incidence. Here we present overview of individual reported cases of nuclear mutations in ATP synthase and discuss, how their analysis can improve our understanding of the enzyme biogenesis.
Physiological research / Academia Scientiarum Bohemoslovaca 02/2014; 63 Suppl 1:S57-71. · 1.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts.
To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-permeabilized cells from 48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with SDS and Blue-Native electrophoreses.
Evaluation of individual respiratory complex activities, ATP synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory complexes I, IV and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed complex I defects; changes in p50 for oxygen and elevated uncoupling control ratio pointed to complex IV deficiency due to SURF1 or SCO2 mutation; high oligomycin sensitivity of state 3 ADP respiration, upregulated mitochondrial membrane potential and low content of complex V were found in lymphocytes with ATP synthase deficiency due to TMEM70 mutations.
Based on our results, we propose the best biochemical parameters predictive for defects of respiratory complexes I, IV and V manifesting in peripheral blood lymphocytes.
The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients.
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 - over 1000 kDa) and cultured cells (400-670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.
PLoS ONE 01/2013; 8(8):e71869. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.
PLoS ONE 01/2013; 8(11):e78627. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.
The FASEB Journal 06/2012; 26(9):3916-30. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The loss of Surf1 protein leads to a severe COX deficiency manifested as a fatal neurodegenerative disorder, the Leigh syndrome (LS(COX)). Surf1 appears to be involved in the early step of COX assembly but its function remains unknown. The aim of the study was to find out how SURF1 gene mutations influence expression of OXPHOS and other pro-mitochondrial genes and to further characterize the altered COX assembly. Analysis of fibroblast cell lines from 9 patients with SURF1 mutations revealed a 70% decrease of the COX complex content to be associated with 32-54% upregulation of respiratory chain complexes I, III and V and accumulation of Cox5a subunit. Whole genome expression profiling showed a general decrease of transcriptional activity in LS(COX) cells and indicated that the adaptive changes in OXPHOS complexes are due to a posttranscriptional compensatory mechanism. Electrophoretic and WB analysis showed that in mitochondria of LS(COX) cells compared to controls, the assembled COX is present entirely in a supercomplex form, as I-III₂-IV supercomplex but not as larger supercomplexes. The lack of COX also caused an accumulation of I-III₂ supercomplex. The accumulated Cox5a was mainly present as a free subunit. We have found out that the major COX assembly subcomplexes accumulated due to SURF1 mutations range in size between approximately 85-140kDa. In addition to the originally proposed S2 intermediate they might also represent Cox1-containing complexes lacking other COX subunits. Unlike the assembled COX, subcomplexes are unable to associate with complexes I and III.
Biochimica et Biophysica Acta 03/2012; 1822(7):1114-24. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Subunit 7a of mouse cytochrome c oxidase (Cox) displays a contractile muscle-specific isoform, Cox7a1, that is the major cardiac form. To gain insight into the role of this isoform, we have produced a new knockout mouse line that lacks Cox7a1. We show that homozygous and heterozygous Cox7a1 knockout mice, although viable, have reduced Cox activity and develop a dilated cardiomyopathy at 6 weeks of age. Surprisingly, the cardiomyopathy improves and stabilizes by 6 months of age. Cox7a1 knockout mice incorporate more of the "liver-type" isoform Cox7a2 into the cardiac Cox holoenzyme and, also surprisingly, have higher tissue ATP levels.
[Show abstract][Hide abstract] ABSTRACT: The primary attempt in diagnostic and experimental studies of numerous pathological states associated with mitochondrial dysfunction is a precise evaluation of changes in function, content and structure of mitochondrial OXPHOS system. The analysis of rat heart, liver, brain and kidney by oxygraphy, enzyme activities, membrane potential, and BN/SDS-PAGE western blotting demonstrated that tissue homogenates can substitute for isolated mitochondria, providing comparable qualitative mitochondrial parameters. The use of homogenate avoids the loss of the majority of mitochondria during their isolation. Only 50-100mg of the tissue is required for the complex OXPHOS analysis, i.e. five times less as compared with isolated mitochondria.
[Show abstract][Hide abstract] ABSTRACT: Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.
[Show abstract][Hide abstract] ABSTRACT: Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.
[Show abstract][Hide abstract] ABSTRACT: Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders.
Biochimica et Biophysica Acta 10/2009; 1802(2):275-83. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of posttranslational modifications, specifically reversible phosphorylation as a regulatory mechanism operating in the mitochondria, is a novel research direction. The mitochondrial oxidative phosphorylation system is a particularly interesting unit because it is responsible for the production of the vast majority of cellular energy in addition to free radicals, two factors that are aberrant in numerous human diseases and that may be influenced by reversible phosphorylation of the oxidative phosphorylation complexes. We here describe a detailed protocol for the isolation of mammalian liver and heart mitochondria and subsequently cytochrome c oxidase (CcO) under conditions maintaining the physiological phosphorylation state. The protocol employs the use of activated vanadate, an unspecific tyrosine phosphatase inhibitor, fluoride, an unspecific serine/threonine phosphatase inhibitor, and EGTA, a calcium chelator to prevent the activation of calcium-dependent protein phosphatases. CcO purified without manipulation of signaling pathways shows strong tyrosine phosphorylation on subunits II and IV, whereas tyrosine phosphorylation of subunit I can be induced by the cAMP- and TNFalpha-dependent pathways in liver. Using our protocol on cow liver tissue we further show the identification of a new phosphorylation site on CcO subunit IV tyrosine 11 of the mature protein (corresponding to tyrosine 33 of the precursor peptide) via immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This phosphorylation site is located close to the ATP and ADP binding site, which adjusts CcO activity to cellular energy demand, and we propose that phosphorylation of tyrosine 11 enables allosteric regulation.
Methods in enzymology 02/2009; 457:193-210. · 1.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of posttranslational modifications, specifically reversible phosphorylation as a regulatory mechanism operating in the mitochondria, is a novel research direction. The mitochondrial oxidative phosphorylation system is a particularly interesting unit because it is responsible for the production of the vast majority of cellular energy in addition to free radicals, two factors that are aberrant in numerous human diseases and that may be influenced by reversible phosphorylation of the oxidative phosphorylation complexes. We here describe a detailed protocol for the isolation of mammalian liver and heart mitochondria and subsequently cytochrome c oxidase (CcO) under conditions maintaining the physiological phosphorylation state. The protocol employs the use of activated vanadate, an unspecific tyrosine phosphatase inhibitor, fluoride, an unspecific serine/threonine phosphatase inhibitor, and EGTA, a calcium chelator to prevent the activation of calcium‐dependent protein phosphatases. CcO purified without manipulation of signaling pathways shows strong tyrosine phosphorylation on subunits II and IV, whereas tyrosine phosphorylation of subunit I can be induced by the cAMP‐ and TNFα‐dependent pathways in liver. Using our protocol on cow liver tissue we further show the identification of a new phosphorylation site on CcO subunit IV tyrosine 11 of the mature protein (corresponding to tyrosine 33 of the precursor peptide) via immobilized metal affinity chromatography/nano‐liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano‐LC/ESI‐MS). This phosphorylation site is located close to the ATP and ADP binding site, which adjusts CcO activity to cellular energy demand, and we propose that phosphorylation of tyrosine 11 enables allosteric regulation.
Methods in Enzymology - METH ENZYMOLOGY. 01/2009; 457:193-210.
[Show abstract][Hide abstract] ABSTRACT: Thirty years after Peter Mitchell was awarded the Nobel Prize for the chemiosmotic hypothesis, which links the mitochondrial membrane potential generated by the proton pumps of the electron transport chain to ATP production by ATP synthase, the molecular players involved once again attract attention. This is so because medical research increasingly recognizes mitochondrial dysfunction as a major factor in the pathology of numerous human diseases, including diabetes, cancer, neurodegenerative diseases, and ischemia reperfusion injury. We propose a model linking mitochondrial oxidative phosphorylation (OxPhos) to human disease, through a lack of energy, excessive free radical production, or a combination of both. We discuss the regulation of OxPhos by cell signaling pathways as a main regulatory mechanism in higher organisms, which in turn determines the magnitude of the mitochondrial membrane potential: if too low, ATP production cannot meet demand, and if too high, free radicals are produced. This model is presented in light of the recently emerging understanding of mechanisms that regulate mammalian cytochrome c oxidase and its substrate cytochrome c as representative enzymes for the entire OxPhos system.
Journal of Bioenergetics 11/2008; 40(5):445-56. · 1.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Recently, the relationship of mitochondrial DNA (mtDNA) variants to metabolic risk factors for diabetes and other common diseases has begun to attract increasing attention. However, progress in this area has been limited because (1) the phenotypic effects of variation in the mitochondrial genome are difficult to isolate owing to confounding variation in the nuclear genome, imprinting phenomena, and environmental factors; and (2) few animal models have been available for directly investigating the effects of mtDNA variants on complex metabolic phenotypes in vivo. Substitution of different mitochondrial genomes on the same nuclear genetic background in conplastic strains provides a way to unambiguously isolate effects of the mitochondrial genome on complex traits. Here we show that conplastic strains of rats with identical nuclear genomes but divergent mitochondrial genomes that encode amino acid differences in proteins of oxidative phosphorylation exhibit differences in major metabolic risk factors for type 2 diabetes. These results (1) provide the first direct evidence linking naturally occurring variation in the mitochondrial genome, independent of variation in the nuclear genome and other confounding factors, to inherited variation in known risk factors for type 2 diabetes; and (2) establish that spontaneous variation in the mitochondrial genome per se can promote systemic metabolic disturbances relevant to the pathogenesis of common diseases.
Genome Research 10/2007; 17(9):1319-26. · 14.40 Impact Factor