Josef Houštěk

TMEM70 mutations cause isolated ATP synthase deficiency and neonatal mitochondrial encephalocardiomyopathy

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.

The function and the role of the mitochondrial glycerol-3-phosphate dehydrogenase in mammalian tissues.

Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is not included in the traditional textbook schemes of the respiratory chain, reflecting the fact that it is a non-standard, tissue-specific component of mammalian mitochondria. But despite its very simple structure, mGPDH is a very important enzyme of intermediary metabolism and as a component of glycerophosphate shuttle it functions at the crossroads of glycolysis, oxidative phosphorylation and fatty acid metabolism. In this review we summarize the present knowledge on the structure and regulation of mGPDH and discuss its metabolic functions, reactive oxygen species production and tissue and organ specific roles in mammalian mitochondria at physiological and pathological conditions.

Mitochondrial ATP synthase deficiency due to a mutation in the ATP5E gene for the F1 ε subunit

F1Fo-ATP synthase is a key enzyme of mitochondrial energy provision producing most of cellular ATP. So far, mitochondrial diseases caused by isolated disorders of the ATP synthase have been shown to result from mutations in mtDNA genes for the subunits ATP6 and ATP8 or in nuclear genes encoding the biogenesis factors TMEM70 and ATPAF2. Here, we describe a patient with a homozygous p.Tyr12Cys mutation in the epsilon subunit encoded by the nuclear gene ATP5E. The 22-year-old woman presented with neonatal onset, lactic acidosis, 3-methylglutaconic aciduria, mild mental retardation and developed peripheral neuropathy. Patient fibroblasts showed 60-70% decrease in both oligomycin-sensitive ATPase activity and mitochondrial ATP synthesis. The mitochondrial content of the ATP synthase complex was equally reduced, but its size was normal and it contained the mutated epsilon subunit. A similar reduction was found in all investigated F1 and Fo subunits with the exception of Fo subunit c, which was found to accumulate in a detergent-insoluble form. This is the first case of a mitochondrial disease due to a mutation in a nuclear encoded structural subunit of the ATP synthase. Our results indicate an essential role of the epsilon subunit in the biosynthesis and assembly of the F1 part of the ATP synthase. Furthermore, the epsilon subunit seems to be involved in the incorporation of subunit c to the rotor structure of the mammalian enzyme.

β‐Adrenergic stimulation of interleukin‐1α and interleukin‐6 expression in mouse brown adipocytes

Mouse brown adipocytes in primary culture were shown to contain high levels of mRNA for interleukin‐1α (IL‐1α) which could be further stimulated up to 9‐fold by norepinephrine (NE). Even higher stimulation by NE, up to 40‐fold, was found in case of interleukin‐6 (IL‐6). Time‐course of activation of both genes was biphasic, but the response of IL‐6 gene was slower than of IL‐1α gene. IL‐1α mRNA level reached the maximum after 1 h and the second, lower increase, occurred after 8 h. IL‐6 mRNA level showed first maximum after 2 h, but the highest level was found after 8 h. Similarly to NE, the expression of IL‐1α and IL‐6 genes was stimulated by selective β‐adrenergic agonist isoproterenol, β3‐selective agonist CGP‐12117, forskoline and db‐cAMP. The activation of both genes by CGP‐12177 was dose‐dependent with the optimum at 100 nM concentration. Stimulation of α‐adrenergic receptors by cirazoline and oxymetazoline was without any effect. When the expression of IL‐6 was studied at the protein level, the stimulation of IL‐6 gene via β3‐receptors resulted in secretion of IL‐6 up to the concentration 10 ng/ml culture media in 24 h. The results indicate a new type of regulation of expression of IL‐1α and IL‐6 genes in brown adipocytes by catecholamines acting via β3‐adrenergic receptors. The resulting increase in IL‐6 production by brown adipocytes could significantly contribute to systemic levels of IL‐6.

Assembly of mitochondrial ATP synthase in cultured human cells: implications for mitochondrial diseases

To study the assembly of mitochondrial F1F0 ATP synthase, cultured human cells were labeled with [35S]methionine in pulse-chase experiments. Next, two-dimensional electrophoresis and fluorography were used to analyze the assembly pattern. Two assembly intermediates could be demonstrated. First the F1 part appeared to be assembled, and next an intermediate product that contained F1 and subunit c. This product probably also contained subunits b, F6 and OSCP, but not the mitochondrially encoded subunits a and A6L. Both intermediate complexes accumulated when mitochondrial protein synthesis was inhibited, suggesting that mitochondrially encoded subunits are indispensable for the formation of a fully assembled ATP synthase complex, but not for the formation of the intermediate complexes. The results and methods described in this study offer an approach to study the effects of mutations in subunits of mitochondrial ATP synthase on the assembly of this complex. This might be of value for a better understanding of deficiencies of ATP synthase activity in mitochrondrial diseases.

Uncoupling protein in embryonic brown adipose tissue — existence of nonthermogenic and thermogenic mitochondria

Embryonic development of mouse and rat brown adipose tissue was characterized by electron microscopy and by quantifying the mitochondrial oxidative, phosphorylating and thermogenic capacities immunochemically, using antibodies against cytochrome oxidase, F1-ATPase and uncoupling protein, respectively. Mitochondria and cytochrome oxidase were detected from the 15-16th day of pregnancy and their amounts continuously increased toward birth. F1-ATPase was also found on the 15th day but it reached a maximum level already on the 19th day when the uncoupling protein appeared and rapidly increased during further maturation of brown adipose tissue. It thus appears that mitochondria in early prenatal brown adipose tissue lack completely uncoupling protein and are nonthermogenic. They transform into typical thermogenic mitochondria abruptly only 2 days before birth.

Tissue Metabolism and Plasma Levels of Thyroid Hormones in Critically Ill Very Premature Infants

Thyroid status was characterized in very preterm infants (gestational age≤32 wk; n = 61) from birth through d 14, and in infants who died within 16 d after delivery (n = 10), where it was also correlated with metabolism of iodothyronines in peripheral tissues (brain, liver, kidney, skeletal muscle, and adipose tissue). At 3 d of life, mean plasma levels of thyroxine, triiodothyronine, and TSH started to decrease, being lower in the critically ill compared with healthy premature neonates. Activities of the three iodothyronine deiodinases enzymes (type I, II, and III, respectively) were detected in all postmortem tissue samples, except for absence of the type II activity in kidney. All activities were the highest in liver and differed in other tissues. Lack of correlation between the type I activity in liver(and kidney), and plasma levels of thyroid hormones suggested that the thyroid was the primary source of circulating triiodothyronine. On the other hand, namely in brain, correlations between activity of the deiodinases and plasma hormone levels were found which suggested a complex control by thyroid hormones of their own metabolism. High activity of type III in liver, adipose tissue, and skeletal muscle demonstrated a role of these tissues in thyroid hormones degradation. Results support the view that peripheral tissues of very preterm infants are engaged in local generation of triiodothyronine, and inactivation of thyroid hormones, but do not represent a major source of circulating triiodothyronine.

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.

High efficiency of ROS production by glycerophosphate dehydrogenase in mammalian mitochondria.

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.

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.