Marek Vrbacky - Academia.edu (original) (raw)

Papers by Marek Vrbacky

Research paper thumbnail of Effects of mtDNA in SHR-mtF344 versus SHR conplastic strains on reduced OXPHOS enzyme levels, insulin resistance, cardiac hypertrophy, and systolic dysfunction

Physiological genomics, Jan 15, 2014

Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtD... more Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtDNA) haplotype groups represented by the BN, F344, LEW, and SHR strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. F344 mtDNA by comparing the SHR vs. SHR-mt(F344) conplastic strains that are genetically identical except for their mitochondrial genomes. Altogether 13 amino acid substitutions in protein coding genes, seven single nucleotide polymorphisms in tRNA genes, and 12 single nucleotide changes in rRNA genes were detected in F344 mtDNA compared with SHR mtDNA. Analysis of oxidative phosphorylation system (OXPHOS) in heart left ventricles (LV), muscle, and liver revealed reduced activity and content of several respiratory chain complexes in SHR-mt(F344) conplastic rats compared with the SHR strain. Lower function of OXPHOS in LV of conplastic rats was associated with significantly increased relative ventricular mass and reduced fractio...

Research paper thumbnail of Mitochondrial ATP synthasome: Expression and structural interactions of its components

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014

ABSTRACT Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (... more ABSTRACT Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (PiC) are supposed to form a supercomplex called ATP synthasome. Our protein and transcript analysis of rat tissues indicates that the expression of ANT and PiC is transcriptionally controlled in accordance with the biogenesis of ATP synthase. In contrast, the content of ANT and PiC is increased in ATP synthase deficient patients' fibroblasts, likely due to a post-transcriptional adaptive mechanism. A structural analysis of rat heart mitochondria by immunoprecipitation, blue native/SDS electrophoresis, immunodetection and MS analysis revealed the presence of ATP synthasome. However, the majority of PiC and especially ANT did not associate with ATP synthase, suggesting that most of PiC, ANT and ATP synthase exist as separate entities. Copyright © 2015. Published by Elsevier Inc.

Research paper thumbnail of Direct linkage of mitochondrial genome variation to risk factors for type 2 diabetes in conplastic strains

Genome Research, 2007

fax (801) 912-3103. Article published online before print. Article and publication date are at

Research paper thumbnail of Tmem70 gene knockout alters biogenesis of ATP synthase and leads to embryonal lethality in mice

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014

Research paper thumbnail of ROS production in brown adipose tissue mitochondria: The question of UCP1-dependence

Keywords: Reactive oxygen species Uncoupling protein 1 Brown adipose tissue mitochondria Cold acc... more Keywords: Reactive oxygen species Uncoupling protein 1 Brown adipose tissue mitochondria Cold acclimation Glycerol-3-phosphate dehydrogenase Succinate Whether active UCP1 can reduce ROS production in brown-fat mitochondria is presently not settled. The issue is of principal significance, as it can be seen as a proof-or disproof-of-principle concerning the ability of any protein to diminish ROS production through membrane depolarization. We therefore undertook a comprehensive investigation of the significance of UCP1 for ROS production, by comparing the ROS production in brown-fat mitochondria isolated from wildtype mice (that display membrane depolarization) or from UCP1(−/−) mice (with a high membrane potential). We tested the significance of UCP1 for glycerol-3-phosphate-supported ROS production by three methods (fluorescent dihydroethidium and the ESR probe PHH for superoxide, and fluorescent Amplex Red for hydrogen peroxide), and followed ROS production also with succinate, acyl-CoA or pyruvate as substrate. We studied the effects of the reverse electron flow inhibitor rotenone, the UCP1 activity inhibitor GDP, and the uncoupler FCCP. We also examined the effect of a physiologically induced increase in UCP1 amount. We noted GDP effects that were not UCP1-related. We conclude that only ROS production supported by exogenously added succinate was affected by the presence of active UCP1; ROS production supported by any other tested substrate (including endogenously generated succinate) was unaffected. This conclusion indicates that UCP1 is not involved in control of ROS production in brown-fat mitochondria. Extrapolation of these data to other tissues would imply that membrane depolarization may not necessarily decrease physiologically relevant ROS production. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Research paper thumbnail of type 2 diabetes in conplastic strains Direct linkage of mitochondrial genome variation to risk factors for

Research paper thumbnail of TMEM70 mutations cause isolated ATP synthase deficiency and neonatal mitochondrial encephalocardiomyopathy

Nature Genetics, 2008

We carried out whole-genome homozygosity mapping, gene expression analysis and DNA sequencing in ... more We carried out whole-genome homozygosity mapping, gene expression analysis and DNA sequencing in individuals with isolated mitochondrial ATP synthase deficiency and identified disease-causing mutations in TMEM70. Complementation of the cell lines of these individuals with wild-type TMEM70 restored biogenesis and metabolic function of the enzyme complex. Our results show that TMEM70 is involved in mitochondrial ATP synthase biogenesis in higher eukaryotes.

Research paper thumbnail of Mitochondrial membrane assembly of TMEM70 protein

Mitochondrion, 2014

Dysfunction of TMEM70 disrupts the biogenesis of ATP synthase and represents the frequent cause o... more Dysfunction of TMEM70 disrupts the biogenesis of ATP synthase and represents the frequent cause of autosomal recessive encephalocardiomyopathy. We used tagged forms of TMEM70 and demonstrated that it has a hairpin structure with the N- and C-termini oriented towards the mitochondrial matrix. On BN-PAGE TMEM70 was detected in multiple forms including dimers and displayed partial overlap with assembled ATP synthase. Immunoprecipitation studies confirmed mutual interactions between TMEM70 molecules but, together with immunogold electron microscopy, not direct interaction with ATP synthase subunits. This indicates that the biological function of TMEM70 in the ATP synthase biogenesis may be mediated through interaction with other protein(s).

Research paper thumbnail of Inhibition of mitochondrial cytochrome c oxidase by dicarbanonaborates

IUBMB Life, 1996

Dicarbanonaborates inhibit the mitochondrial cytochrome c oxidase activity. In contrast to mitoch... more Dicarbanonaborates inhibit the mitochondrial cytochrome c oxidase activity. In contrast to mitochondrial ATPase or glycerol phosphate dehydrogenase, inhibition of cytochrome c oxidase was not competitive and the residual, drug-insensitive activity was higher. These results indicate that dicarbanonaborates inhibit various mitochondrial membrane-bound enzymes through different mechanisms.

Research paper thumbnail of Molecular mechanisms of silybin and 2,3-dehydrosilybin antiradical activity—role of individual hydroxyl groups

Free Radical Biology and Medicine, 2009

The flavonolignans silybin (1) and 2,3-dehydrosilybin (2) are important natural compounds with mu... more The flavonolignans silybin (1) and 2,3-dehydrosilybin (2) are important natural compounds with multiple biological activities operating at various cell levels. Many of these effects are connected with their radical-scavenging activities. The molecular mechanisms of the antioxidant activity of these compounds and even the functional groups responsible for this activity are not yet well known. Their mechanism can be inferred from the structures of the dimeric products obtained from radical-mediated reactions of selectively methylated derivatives of 1 and 2. The radical oxidation of 1 methylated at 7-OH and 2 methylated at both 3-OH and 7-OH yields C-C and C-O dimers that enable the molecular mechanism of their E-ring interaction with radicals to be elucidated and shows the importance of the 20-OH group in this respect. The pivotal role of the 3-OH group in the radical-scavenging activity of 2 was confirmed through the formation of another type of dimer from its selectively methylated derivative.

Research paper thumbnail of Effect of metformin therapy on cardiac function and survival in a volume-overload model of heart failure in rats

Clinical Science, 2011

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification... more Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg · kg − 1 of body weight · day − 1 , 0.5 % in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n = 6-12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n = 10-90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified 'free' fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial 14 C-glucose and 14 C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2 + − 0.7 μg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF.

Research paper thumbnail of Molecular Characterization of Binding of Calcium and Carbohydrates by an Early Activation Antigen of Lymphocytes CD69 †

Biochemistry, 2003

CD69 is the earliest leukocyte activation antigen playing a pivotal role in cellular signaling. H... more CD69 is the earliest leukocyte activation antigen playing a pivotal role in cellular signaling. Here, we show that a globular C-terminal domain of CD69 belonging to C-type lectins binds calcium through Asp 171, Glu 185, and Glu 187 with K d ∼54 µM. Closure of the calcium-binding site results in a conformational shift of Thr 107 and Lys 172. Interestingly, structural changes in all of these amino acids lead to the formation of high-affinity binding sites for N-acetyl-D-glucosamine. Similarly, a structural change in Glu 185 and Glu 187 contributes to a high-affinity site for N-acetyl-D-galactosamine. Sitedirected mutagenesis and molecular modeling allowed us to describe the structural details of binding sites for both carbohydrates. These studies explain the importance of calcium for recognition of carbohydrates by CD69 and provide an important paradigm for the role of weak interactions in the immune system.

Research paper thumbnail of Succinimidyl oleate, established inhibitor of CD36/FAT translocase inhibits complex III of mitochondrial respiratory chain

Biochemical and Biophysical Research Communications, 2010

The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid ... more The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid (LCFA) oxidation is unclear due to conflicting results obtained in Cd36 knockout mice and experiments using sulfo-N-succinimidyl oleate (SSO) for inhibition of CD36 mediated LCFA transport. We investigated effect of SSO on mitochondrial respiration and found that SSO substantially inhibits not only LCFA oxidation, but also oxidation of flavoprotein- and NADH-dependent substrates and generation of mitochondrial membrane potential. Experiments in rat liver, heart and kidney mitochondria demonstrated a direct effect on mitochondrial respiratory chain with the most pronounced inhibition of the complex III (IC(50) 4microM SSO). The results presented here show that SSO is a potent and irreversible inhibitor of mitochondrial respiratory chain.

Research paper thumbnail of Inhibition of glycerophosphate-dependent H2O2 generation in brown fat mitochondria by idebenone

Biochemical and Biophysical Research Communications, 2006

The established protective effect of coenzyme Q (CoQ) analogs is dependent on the location of rea... more The established protective effect of coenzyme Q (CoQ) analogs is dependent on the location of reactive oxygen species (ROS) generation. One of these analogs--idebenone (hydroxydecyl-ubiquinone) is used as an antioxidative therapeutic drug. We tested its scavenging effect on the glycerophosphate (GP)-dependent ROS production as this enzyme was shown as a new site in the mitochondrial respiratory chain where ROS can be generated. We observed that idebenone inhibits both GP- and succinate-dependent ROS production. Idebenone and CoQ1 were found to be more efficient in the scavenging activity (IC50: 0.052 and 0.075 microM, respectively) than CoQ3 (IC50: 45.8 microM). Idebenone also inhibited ferricyanide (FeCN)-activated, GP-dependent ROS production. Our data thus extend previous findings on the scavenging effect of idebenone and show that it can also eliminate GP-dependent ROS generation.

Research paper thumbnail of Expression and processing of the TMEM70 protein

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2011

a b s t r a c t TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We ... more a b s t r a c t TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We have investigated import and processing of this factor in human cells using GFP-and FLAG-tagged forms of TMEM70 and specific antibodies. TMEM70 is synthesized as a 29 kDa precursor protein that is processed to a 21 kDa mature form. Immunocytochemical detection of TMEM70 showed mitochondrial colocalization with MitoTracker Red and ATP synthase. Western blot of subcellular fractions revealed the highest signal of TMEM70 in isolated mitochondria and mitochondrial location was confirmed by mass spectrometry analysis. Based on analysis of submitochondrial fractions, TMEM70 appears to be located in the inner mitochondrial membrane, in accordance with predicated transmembrane regions in the central part of the TMEM70 sequence. Twodimensional electrophoretic analysis did not show direct interaction of TMEM70 with assembled ATP synthase but indicated the presence of dimeric form of TMEM70. No TMEM70 protein could be found in cells and isolated mitochondria from patients with ATP synthase deficiency due to TMEM70 c.317-2ANG mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.

Research paper thumbnail of Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2007

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in rea... more Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H 2 DCFDA and Amplex Red was used to determine the glycerophosphate-or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphatedependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.

Research paper thumbnail of Cyanide inhibition and pyruvate-induced recovery of cytochrome c oxidase

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010

The mechanism of cyanide's inhibitory effect on the mitochondrial... more The mechanism of cyanide's inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ(m)) and the enzyme affinity to oxygen (p₅₀ value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60-80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects.

Research paper thumbnail of High efficiency of ROS production by glycerophosphate dehydrogenase in mammalian mitochondria

Archives of Biochemistry and Biophysics, 2009

We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and h... more We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and high (brown adipose tissue, BAT) content of glycerophosphate dehydrogenase (mGPDH). ROS production at state 4 due to electron backflow from mGPDH was low, but after inhibition of electron transport with antimycin A high rates of mGPDH-dependent ROS production were observed in liver, heart and brain mitochondria. When this ROS production was related to activity of mGPDH, many-fold higher ROS production was found in contrast to succinate- (39-, 28-, 3-fold) or pyruvate plus malate-dependent ROS production (32-, 96-, 5-fold). This specific rate of mGPDH-dependent ROS production was also exceedingly higher (28-, 66-, 22-fold) compared to that in BAT. mGPDH-dependent ROS production was localized to the dehydrogenase+CoQ and complex III, the latter being the highest in all mitochondria but BAT. Our results demonstrate high efficiency of mGPDH-dependent ROS production in mammalian mitochondria with a low content of mGPDH and suggest its endogenous inhibition in BAT.

Research paper thumbnail of Uncoupling protein-1 is not leaky

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010

Uncoupling protein 1 Brown adipose tissue mitochondria Cold acclimation Thermogenesis Basal proto... more Uncoupling protein 1 Brown adipose tissue mitochondria Cold acclimation Thermogenesis Basal proton leak Medium tonicity

Research paper thumbnail of Effects of mtDNA in SHR-mtF344 versus SHR conplastic strains on reduced OXPHOS enzyme levels, insulin resistance, cardiac hypertrophy, and systolic dysfunction

Physiological genomics, Jan 15, 2014

Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtD... more Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtDNA) haplotype groups represented by the BN, F344, LEW, and SHR strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. F344 mtDNA by comparing the SHR vs. SHR-mt(F344) conplastic strains that are genetically identical except for their mitochondrial genomes. Altogether 13 amino acid substitutions in protein coding genes, seven single nucleotide polymorphisms in tRNA genes, and 12 single nucleotide changes in rRNA genes were detected in F344 mtDNA compared with SHR mtDNA. Analysis of oxidative phosphorylation system (OXPHOS) in heart left ventricles (LV), muscle, and liver revealed reduced activity and content of several respiratory chain complexes in SHR-mt(F344) conplastic rats compared with the SHR strain. Lower function of OXPHOS in LV of conplastic rats was associated with significantly increased relative ventricular mass and reduced fractio...

Research paper thumbnail of Mitochondrial ATP synthasome: Expression and structural interactions of its components

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014

ABSTRACT Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (... more ABSTRACT Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (PiC) are supposed to form a supercomplex called ATP synthasome. Our protein and transcript analysis of rat tissues indicates that the expression of ANT and PiC is transcriptionally controlled in accordance with the biogenesis of ATP synthase. In contrast, the content of ANT and PiC is increased in ATP synthase deficient patients' fibroblasts, likely due to a post-transcriptional adaptive mechanism. A structural analysis of rat heart mitochondria by immunoprecipitation, blue native/SDS electrophoresis, immunodetection and MS analysis revealed the presence of ATP synthasome. However, the majority of PiC and especially ANT did not associate with ATP synthase, suggesting that most of PiC, ANT and ATP synthase exist as separate entities. Copyright © 2015. Published by Elsevier Inc.

Research paper thumbnail of Direct linkage of mitochondrial genome variation to risk factors for type 2 diabetes in conplastic strains

Genome Research, 2007

fax (801) 912-3103. Article published online before print. Article and publication date are at

Research paper thumbnail of Tmem70 gene knockout alters biogenesis of ATP synthase and leads to embryonal lethality in mice

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014

Research paper thumbnail of ROS production in brown adipose tissue mitochondria: The question of UCP1-dependence

Keywords: Reactive oxygen species Uncoupling protein 1 Brown adipose tissue mitochondria Cold acc... more Keywords: Reactive oxygen species Uncoupling protein 1 Brown adipose tissue mitochondria Cold acclimation Glycerol-3-phosphate dehydrogenase Succinate Whether active UCP1 can reduce ROS production in brown-fat mitochondria is presently not settled. The issue is of principal significance, as it can be seen as a proof-or disproof-of-principle concerning the ability of any protein to diminish ROS production through membrane depolarization. We therefore undertook a comprehensive investigation of the significance of UCP1 for ROS production, by comparing the ROS production in brown-fat mitochondria isolated from wildtype mice (that display membrane depolarization) or from UCP1(−/−) mice (with a high membrane potential). We tested the significance of UCP1 for glycerol-3-phosphate-supported ROS production by three methods (fluorescent dihydroethidium and the ESR probe PHH for superoxide, and fluorescent Amplex Red for hydrogen peroxide), and followed ROS production also with succinate, acyl-CoA or pyruvate as substrate. We studied the effects of the reverse electron flow inhibitor rotenone, the UCP1 activity inhibitor GDP, and the uncoupler FCCP. We also examined the effect of a physiologically induced increase in UCP1 amount. We noted GDP effects that were not UCP1-related. We conclude that only ROS production supported by exogenously added succinate was affected by the presence of active UCP1; ROS production supported by any other tested substrate (including endogenously generated succinate) was unaffected. This conclusion indicates that UCP1 is not involved in control of ROS production in brown-fat mitochondria. Extrapolation of these data to other tissues would imply that membrane depolarization may not necessarily decrease physiologically relevant ROS production. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Research paper thumbnail of type 2 diabetes in conplastic strains Direct linkage of mitochondrial genome variation to risk factors for

Research paper thumbnail of TMEM70 mutations cause isolated ATP synthase deficiency and neonatal mitochondrial encephalocardiomyopathy

Nature Genetics, 2008

We carried out whole-genome homozygosity mapping, gene expression analysis and DNA sequencing in ... more We carried out whole-genome homozygosity mapping, gene expression analysis and DNA sequencing in individuals with isolated mitochondrial ATP synthase deficiency and identified disease-causing mutations in TMEM70. Complementation of the cell lines of these individuals with wild-type TMEM70 restored biogenesis and metabolic function of the enzyme complex. Our results show that TMEM70 is involved in mitochondrial ATP synthase biogenesis in higher eukaryotes.

Research paper thumbnail of Mitochondrial membrane assembly of TMEM70 protein

Mitochondrion, 2014

Dysfunction of TMEM70 disrupts the biogenesis of ATP synthase and represents the frequent cause o... more Dysfunction of TMEM70 disrupts the biogenesis of ATP synthase and represents the frequent cause of autosomal recessive encephalocardiomyopathy. We used tagged forms of TMEM70 and demonstrated that it has a hairpin structure with the N- and C-termini oriented towards the mitochondrial matrix. On BN-PAGE TMEM70 was detected in multiple forms including dimers and displayed partial overlap with assembled ATP synthase. Immunoprecipitation studies confirmed mutual interactions between TMEM70 molecules but, together with immunogold electron microscopy, not direct interaction with ATP synthase subunits. This indicates that the biological function of TMEM70 in the ATP synthase biogenesis may be mediated through interaction with other protein(s).

Research paper thumbnail of Inhibition of mitochondrial cytochrome c oxidase by dicarbanonaborates

IUBMB Life, 1996

Dicarbanonaborates inhibit the mitochondrial cytochrome c oxidase activity. In contrast to mitoch... more Dicarbanonaborates inhibit the mitochondrial cytochrome c oxidase activity. In contrast to mitochondrial ATPase or glycerol phosphate dehydrogenase, inhibition of cytochrome c oxidase was not competitive and the residual, drug-insensitive activity was higher. These results indicate that dicarbanonaborates inhibit various mitochondrial membrane-bound enzymes through different mechanisms.

Research paper thumbnail of Molecular mechanisms of silybin and 2,3-dehydrosilybin antiradical activity—role of individual hydroxyl groups

Free Radical Biology and Medicine, 2009

The flavonolignans silybin (1) and 2,3-dehydrosilybin (2) are important natural compounds with mu... more The flavonolignans silybin (1) and 2,3-dehydrosilybin (2) are important natural compounds with multiple biological activities operating at various cell levels. Many of these effects are connected with their radical-scavenging activities. The molecular mechanisms of the antioxidant activity of these compounds and even the functional groups responsible for this activity are not yet well known. Their mechanism can be inferred from the structures of the dimeric products obtained from radical-mediated reactions of selectively methylated derivatives of 1 and 2. The radical oxidation of 1 methylated at 7-OH and 2 methylated at both 3-OH and 7-OH yields C-C and C-O dimers that enable the molecular mechanism of their E-ring interaction with radicals to be elucidated and shows the importance of the 20-OH group in this respect. The pivotal role of the 3-OH group in the radical-scavenging activity of 2 was confirmed through the formation of another type of dimer from its selectively methylated derivative.

Research paper thumbnail of Effect of metformin therapy on cardiac function and survival in a volume-overload model of heart failure in rats

Clinical Science, 2011

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification... more Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg · kg − 1 of body weight · day − 1 , 0.5 % in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n = 6-12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n = 10-90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified 'free' fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial 14 C-glucose and 14 C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2 + − 0.7 μg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF.

Research paper thumbnail of Molecular Characterization of Binding of Calcium and Carbohydrates by an Early Activation Antigen of Lymphocytes CD69 †

Biochemistry, 2003

CD69 is the earliest leukocyte activation antigen playing a pivotal role in cellular signaling. H... more CD69 is the earliest leukocyte activation antigen playing a pivotal role in cellular signaling. Here, we show that a globular C-terminal domain of CD69 belonging to C-type lectins binds calcium through Asp 171, Glu 185, and Glu 187 with K d ∼54 µM. Closure of the calcium-binding site results in a conformational shift of Thr 107 and Lys 172. Interestingly, structural changes in all of these amino acids lead to the formation of high-affinity binding sites for N-acetyl-D-glucosamine. Similarly, a structural change in Glu 185 and Glu 187 contributes to a high-affinity site for N-acetyl-D-galactosamine. Sitedirected mutagenesis and molecular modeling allowed us to describe the structural details of binding sites for both carbohydrates. These studies explain the importance of calcium for recognition of carbohydrates by CD69 and provide an important paradigm for the role of weak interactions in the immune system.

Research paper thumbnail of Succinimidyl oleate, established inhibitor of CD36/FAT translocase inhibits complex III of mitochondrial respiratory chain

Biochemical and Biophysical Research Communications, 2010

The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid ... more The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid (LCFA) oxidation is unclear due to conflicting results obtained in Cd36 knockout mice and experiments using sulfo-N-succinimidyl oleate (SSO) for inhibition of CD36 mediated LCFA transport. We investigated effect of SSO on mitochondrial respiration and found that SSO substantially inhibits not only LCFA oxidation, but also oxidation of flavoprotein- and NADH-dependent substrates and generation of mitochondrial membrane potential. Experiments in rat liver, heart and kidney mitochondria demonstrated a direct effect on mitochondrial respiratory chain with the most pronounced inhibition of the complex III (IC(50) 4microM SSO). The results presented here show that SSO is a potent and irreversible inhibitor of mitochondrial respiratory chain.

Research paper thumbnail of Inhibition of glycerophosphate-dependent H2O2 generation in brown fat mitochondria by idebenone

Biochemical and Biophysical Research Communications, 2006

The established protective effect of coenzyme Q (CoQ) analogs is dependent on the location of rea... more The established protective effect of coenzyme Q (CoQ) analogs is dependent on the location of reactive oxygen species (ROS) generation. One of these analogs--idebenone (hydroxydecyl-ubiquinone) is used as an antioxidative therapeutic drug. We tested its scavenging effect on the glycerophosphate (GP)-dependent ROS production as this enzyme was shown as a new site in the mitochondrial respiratory chain where ROS can be generated. We observed that idebenone inhibits both GP- and succinate-dependent ROS production. Idebenone and CoQ1 were found to be more efficient in the scavenging activity (IC50: 0.052 and 0.075 microM, respectively) than CoQ3 (IC50: 45.8 microM). Idebenone also inhibited ferricyanide (FeCN)-activated, GP-dependent ROS production. Our data thus extend previous findings on the scavenging effect of idebenone and show that it can also eliminate GP-dependent ROS generation.

Research paper thumbnail of Expression and processing of the TMEM70 protein

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2011

a b s t r a c t TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We ... more a b s t r a c t TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We have investigated import and processing of this factor in human cells using GFP-and FLAG-tagged forms of TMEM70 and specific antibodies. TMEM70 is synthesized as a 29 kDa precursor protein that is processed to a 21 kDa mature form. Immunocytochemical detection of TMEM70 showed mitochondrial colocalization with MitoTracker Red and ATP synthase. Western blot of subcellular fractions revealed the highest signal of TMEM70 in isolated mitochondria and mitochondrial location was confirmed by mass spectrometry analysis. Based on analysis of submitochondrial fractions, TMEM70 appears to be located in the inner mitochondrial membrane, in accordance with predicated transmembrane regions in the central part of the TMEM70 sequence. Twodimensional electrophoretic analysis did not show direct interaction of TMEM70 with assembled ATP synthase but indicated the presence of dimeric form of TMEM70. No TMEM70 protein could be found in cells and isolated mitochondria from patients with ATP synthase deficiency due to TMEM70 c.317-2ANG mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.

Research paper thumbnail of Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2007

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in rea... more Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H 2 DCFDA and Amplex Red was used to determine the glycerophosphate-or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphatedependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.

Research paper thumbnail of Cyanide inhibition and pyruvate-induced recovery of cytochrome c oxidase

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010

The mechanism of cyanide's inhibitory effect on the mitochondrial... more The mechanism of cyanide's inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ(m)) and the enzyme affinity to oxygen (p₅₀ value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60-80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects.

Research paper thumbnail of High efficiency of ROS production by glycerophosphate dehydrogenase in mammalian mitochondria

Archives of Biochemistry and Biophysics, 2009

We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and h... more We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and high (brown adipose tissue, BAT) content of glycerophosphate dehydrogenase (mGPDH). ROS production at state 4 due to electron backflow from mGPDH was low, but after inhibition of electron transport with antimycin A high rates of mGPDH-dependent ROS production were observed in liver, heart and brain mitochondria. When this ROS production was related to activity of mGPDH, many-fold higher ROS production was found in contrast to succinate- (39-, 28-, 3-fold) or pyruvate plus malate-dependent ROS production (32-, 96-, 5-fold). This specific rate of mGPDH-dependent ROS production was also exceedingly higher (28-, 66-, 22-fold) compared to that in BAT. mGPDH-dependent ROS production was localized to the dehydrogenase+CoQ and complex III, the latter being the highest in all mitochondria but BAT. Our results demonstrate high efficiency of mGPDH-dependent ROS production in mammalian mitochondria with a low content of mGPDH and suggest its endogenous inhibition in BAT.

Research paper thumbnail of Uncoupling protein-1 is not leaky

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010

Uncoupling protein 1 Brown adipose tissue mitochondria Cold acclimation Thermogenesis Basal proto... more Uncoupling protein 1 Brown adipose tissue mitochondria Cold acclimation Thermogenesis Basal proton leak Medium tonicity