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 29kDa precursor protein that is processed to a 21kDa 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. Two-dimensional 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.317-2A>G mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.

• MeSH
• buněčné linie MeSH
• Escherichia coli enzymologie   MeSH
• fibroblasty enzymologie   MeSH
• hmotnostní spektrometrie metody   MeSH
• klonování DNA MeSH
• komplementární DNA genetika   MeSH
• ledviny enzymologie   MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• mitochondriální proteiny chemie   genetika   metabolismus   MeSH
• mitochondriální protonové ATPasy nedostatek   MeSH
• mitochondrie enzymologie   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• skot MeSH
• submitochondriální částice enzymologie   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Klíčová slova
• TMEM70, komplex V, ATPsyntáza,
• MeSH
• biogeneze organel MeSH
• buněčné linie MeSH
• energetický metabolismus fyziologie   MeSH
• fumarasa aplikace a dávkování   MeSH
• lidé MeSH
• mapování interakce mezi proteiny metody   využití   MeSH
• membránové proteiny genetika   chemie   metabolismus   MeSH
• mitochondriální ADP/ATP-translokasy MeSH
• mitochondriální proteiny biosyntéza   nedostatek   MeSH
• mitochondriální protonové ATPasy genetika   nedostatek   MeSH
• mitochondrie * enzymologie   MeSH
• oxidativní fosforylace MeSH
• prospektivní studie MeSH
• transportní proteiny mitochondriální membrány MeSH
• trypsin aplikace a dávkování   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

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).

• MeSH
• buněčné linie MeSH
• imunoelektronová mikroskopie MeSH
• imunoprecipitace MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondriální protonové ATPasy metabolismus   MeSH
• multimerizace proteinu * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Klíčová slova
• 3-methylglutakonová acidurie, 3-methylglutakonová acidurie,
• MeSH
• acidóza laktátová epidemiologie   prevence a kontrola   terapie   MeSH
• anoctamin 1 * genetika   nedostatek   MeSH
• dietoterapie normy   MeSH
• dítě MeSH
• etnicita MeSH
• gastrostomie MeSH
• glutaráty metabolismus   moč   MeSH
• hydrogenuhličitany terapeutické užití   MeSH
• hypospadie MeSH
• inguinální hernie MeSH
• komplikace těhotenství MeSH
• kraniofaciální abnormality MeSH
• lidé MeSH
• mitochondriální nemoci genetika   MeSH
• oligohydramnion MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH
• práce podpořená grantem MeSH

OBJECTIVES: TMEM70 deficiency is the most common nuclear-encoded defect affecting the ATP synthase. In this multicentre retrospective study we characterise the natural history of the disease, treatment and outcome in 48 patients with mutations in TMEM70. Eleven centers from eight European countries, Turkey and Israel participated. RESULTS: All 27 Roma and eight non-Roma patients were homozygous for the common mutation c.317-2A > G. Five patients were compound heterozygotes for the common mutation and mutations c.470 T > A, c.628A > C, c.118_119insGT or c.251delC. Six Arab Muslims and two Turkish patients were homozygous for mutations c.238C > T, c.316 + 1G > T, c.336 T > A, c.578_579delCA, c.535C > T, c.359delC. Age of onset was neonatal in 41 patients, infantile in six cases and two years in one child. The most frequent symptoms at onset were poor feeding, hypotonia, lethargy, respiratory and heart failure, accompanied by lactic acidosis, 3-methylglutaconic aciduria and hyperammonaemia. Symptoms further included: developmental delay (98%), hypotonia (95%), faltering growth (94%), short stature (89%), non-progressive cardiomyopathy (89%), microcephaly (71%), facial dysmorphism (66%), hypospadias (50% of the males), persistent pulmonary hypertension of the newborn (22%) and Wolff-Parkinson-White syndrome (13%). One or more acute metabolic crises occurred in 24 surviving children, frequently followed by developmental regression. Hyperammonaemic episodes responded well to infusion with glucose and lipid emulsion, and ammonia scavengers or haemodiafiltration. Ten-year survival was 63%, importantly for prognostication, no child died after the age of five years. CONCLUSION: TMEM70 deficiency is a panethnic, multisystemic disease with variable outcome depending mainly on adequate management of hyperammonaemic crises in the neonatal period and early childhood.

• MeSH
• acidóza laktátová genetika   MeSH
• dítě MeSH
• dospělí MeSH
• heterozygot MeSH
• homozygot MeSH
• hyperamonemie genetika   MeSH
• Kaplanův-Meierův odhad MeSH
• kardiomyopatie genetika   MeSH
• kojenec MeSH
• kosterní svaly patologie   MeSH
• lidé MeSH
• management nemoci MeSH
• membránové proteiny nedostatek   genetika   MeSH
• mitochondriální proteiny nedostatek   genetika   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutace MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• retrospektivní studie MeSH
• vrozené poruchy metabolismu genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH
• Izrael MeSH
• Turecko MeSH

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.

• MeSH
• buněčné linie MeSH
• financování organizované MeSH
• kardiomyopatie enzymologie   genetika   komplikace   MeSH
• klonování DNA MeSH
• komplementární DNA genetika   MeSH
• lidé MeSH
• membránové proteiny genetika   MeSH
• mitochondriální encefalomyopatie enzymologie   genetika   komplikace   MeSH
• mitochondriální proteiny genetika   MeSH
• mitochondriální protonové ATPasy nedostatek   MeSH
• mutace genetika   MeSH
• novorozenec MeSH
• testy genetické komplementace MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH

An increasing number of patients with nuclear genetic defects of mitochondrial ATP synthase have been identified in recent years. They are characterized by early onset, lactic acidosis, 3-methylglutaconic aciduria, hypertrophic cardiomyopathy and encephalopathy and most cases have a fatal outcome. Patient tissues show isolated defect of the ATP synthase complex and its content decreases to > or =30% of normal due to altered enzyme biosynthesis and assembly. Gene mapping and complementation studies have identified mutations in TMEM70 gene encoding a 30kD mitochondrial protein of unknown function as the cause of the disease. An altered synthesis of this new ancillary factor in ATP synthase biogenesis was found in most of the known patients with decreased ATP synthase content. As revealed by phylogenetic analysis, TMEM70 is specific for higher eukaryotes.

• MeSH
• buněčné jádro genetika   patologie   MeSH
• druhová specificita MeSH
• familiární hypertrofická kardiomyopatie genetika   metabolismus   MeSH
• fylogeneze MeSH
• lidé MeSH
• membránové proteiny * genetika   MeSH
• mitochondriální proteiny * genetika   MeSH
• mitochondriální protonové ATPasy genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• mutace MeSH
• nemoci mozku genetika   metabolismus   MeSH
• oxidativní fosforylace MeSH
• respirační komplex IV genetika   metabolismus   MeSH
• savci MeSH
• testy genetické komplementace MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

OBJECTIVE: Mitochondrial disturbances of energygenerating systems in childhood are a heterogeneous group of disorders. The aim of this multi-site survey was to characterise the natural course of a novel mitochondrial disease with ATP synthase deficiency and mutation in the TMEM70 gene. METHODS: Retrospective clinical data and metabolic profiles were collected and evaluated in 25 patients (14 boys, 11 girls) from seven European countries with a c.317-2A-->G mutation in the TMEM70 gene. RESULTS: Severe muscular hypotonia (in 92% of newborns), apnoic spells (92%), hypertrophic cardiomyopathy (HCMP; 76%) and profound lactic acidosis (lactate 5-36 mmol/l; 92%) with hyperammonaemia (100-520 micromol/l; 86%) were present from birth. Ten patients died within the first 6 weeks of life. Most patients surviving the neonatal period had persisting muscular hypotonia and developed psychomotor delay. HCMP was non-progressive and even disappeared in some children. Hypospadia was present in 54% of the boys and cryptorchidism in 67%. Increased excretion of lactate and 3-methylglutaconic acid (3-MGC) was observed in all patients. In four surviving patients, life-threatening hyperammonaemia occurred during childhood, triggered by acute gastroenteritis and prolonged fasting. CONCLUSIONS: ATP synthase deficiency with mutation in TMEM70 should be considered in the diagnosis and management of critically ill neonates with early neonatal onset of muscular hypotonia, HCMP and hypospadias in boys accompanied by lactic acidosis, hyperammonaemia and 3-MGC-uria. However, phenotype severity may vary significantly. The disease occurs frequently in the Roma population and molecular-genetic analysis of the TMEM70 gene is sufficient for diagnosis without need of muscle biopsy in affected children.

• MeSH
• fenotyp MeSH
• hyperamonemie enzymologie   genetika   MeSH
• hypospadie enzymologie   genetika   MeSH
• kojenec MeSH
• kryptorchismus enzymologie   genetika   MeSH
• lidé MeSH
• membránové proteiny genetika   MeSH
• mitochondriální encefalomyopatie enzymologie   genetika   MeSH
• mitochondriální proteiny genetika   MeSH
• mitochondriální protonové ATPasy nedostatek   MeSH
• mutace MeSH
• novorozenec MeSH
• retrospektivní studie MeSH
• věk při počátku nemoci MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH

TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• homozygot MeSH
• kardiomyopatie metabolismus   MeSH
• membránové proteiny nedostatek   genetika   metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondriální nemoci genetika   metabolismus   MeSH
• mitochondriální proteiny nedostatek   genetika   metabolismus   MeSH
• mitochondriální protonové ATPasy biosyntéza   genetika   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• mutace MeSH
• myši knockoutované MeSH
• myši MeSH
• oxidativní fosforylace MeSH
• těhotenství MeSH
• vrozené poruchy metabolismu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Klíčová slova
• mitochindrie, ATPsyntáza, biogeneze,
• MeSH
• financování organizované MeSH
• lidé MeSH
• mitochondriální nemoci genetika   MeSH
• mitochondriální protonové ATPasy genetika   metabolismus   MeSH
• transportní proteiny mitochondriální membrány biosyntéza   genetika   MeSH
• Check Tag
• lidé MeSH