[Show abstract][Hide abstract] ABSTRACT: The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.
Frontiers in Genetics 02/2015; 6:21. DOI:10.3389/fgene.2015.00021
[Show abstract][Hide abstract] ABSTRACT: Objective:
We report novel defects of mitochondrial translation elongation factor Ts (EFTs), with high carrier frequency in Finland and expand the manifestations of this disease group from infantile cardiomyopathy to juvenile neuropathy/encephalopathy disorders.
DNA analysis, whole-exome analysis, protein biochemistry, and protein modeling.
We used whole-exome sequencing to find the genetic cause of infantile-onset mitochondrial cardiomyopathy, progressing to juvenile-onset Leigh syndrome, neuropathy, and optic atrophy in 2 siblings. We found novel compound heterozygous mutations, c.944G>A [p.C315Y] and c.856C>T [p.Q286X], in the TSFM gene encoding mitochondrial EFTs. The same p.Q286X variant was found as compound heterozygous with a splice site change in a patient from a second family, with juvenile-onset optic atrophy, peripheral neuropathy, and ataxia. Our molecular modeling predicted the coding-region mutations to cause protein instability, which was experimentally confirmed in cultured patient cells, with mitochondrial translation defect and lacking EFTs. Only a single TSFM mutation has been previously described in different populations, leading to an infantile fatal multisystem disorder with cardiomyopathy. Sequence data from 35,000 Finnish population controls indicated that the heterozygous carrier frequency of p.Q286X change was exceptionally high in Finland, 1:80, but no homozygotes were found in the population, in our mitochondrial disease patient collection, or in an intrauterine fetal death material, suggesting early developmental lethality of the homozygotes.
We show that in addition to early-onset cardiomyopathy, TSFM mutations should be considered in childhood and juvenile encephalopathies with optic and/or peripheral neuropathy, ataxia, or Leigh disease.
[Show abstract][Hide abstract] ABSTRACT: Mutations in SUCLA2, encoding the ß-subunit of succinyl-CoA synthetase of Krebs cycle, are one cause of mitochondrial DNA depletion syndrome. Patients have been reported to have severe progressive childhood-onset encephalomyopathy, and methylmalonic aciduria, often leading to death in childhood. We studied two families, with children manifesting with slowly progressive mitochondrial encephalomyopathy, hearing impairment and transient methylmalonic aciduria, without mtDNA depletion. The other family also showed dominant inheritance of bilateral retinoblastoma, which coexisted with mitochondrial encephalomyopathy in one patient. We found a variant in SUCLA2 leading to Asp333Gly change, homozygous in one patient and compound heterozygous in one. The latter patient also carried a deletion of 13q14 of the other allele, discovered with molecular karyotyping. The deletion spanned both SUCLA2 and RB1 gene regions, leading to manifestation of both mitochondrial disease and retinoblastoma. We made a homology model for human succinyl-CoA synthetase and used it for structure-function analysis of all reported pathogenic mutations in SUCLA2. On the basis of our model, all previously described mutations were predicted to result in decreased amounts of incorrectly assembled protein or disruption of ADP phosphorylation, explaining the severe early lethal manifestations. However, the Asp333Gly change was predicted to reduce the activity of the otherwise functional enzyme. On the basis of our findings, SUCLA2 mutations should be analyzed in patients with slowly progressive encephalomyopathy, even in the absence of methylmalonic aciduria or mitochondrial DNA depletion. In addition, an encephalomyopathy in a patient with retinoblastoma suggests mutations affecting SUCLA2.European Journal of Human Genetics advance online publication, 2 July 2014; doi:10.1038/ejhg.2014.128.
European journal of human genetics: EJHG 07/2014; 23(3). DOI:10.1038/ejhg.2014.128 · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nutrient availability is the major regulator of life and reproduction, and a complex cellular signaling network has evolved to adapt organisms to fasting. These sensor pathways monitor cellular energy metabolism, especially mitochondrial ATP production and NAD+/NADH ratio, as major signals for nutritional state. We hypothesized that these signals would be modified by mitochondrial respiratory chain disease, because of inefficient NADH utilization and ATP production. Oral administration of nicotinamide riboside (NR), a vitamin B3 and NAD+ precursor, was previously shown to boost NAD+ levels in mice and to induce mitochondrial biogenesis. Here, we treated mitochondrial myopathy mice with NR. This vitamin effectively delayed early- and late-stage disease progression, by robustly inducing mitochondrial biogenesis in skeletal muscle and brown adipose tissue, preventing mitochondrial ultrastructure abnormalities and mtDNA deletion formation. NR further stimulated mitochondrial unfolded protein response, suggesting its protective role in mitochondrial disease. These results indicate that NR and strategies boosting NAD+ levels are a promising treatment strategy for mitochondrial myopathy.
EMBO Molecular Medicine 04/2014; 6(6). DOI:10.1002/emmm.201403943 · 8.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
The genetic complexity of infantile cardiomyopathies is remarkable, and the importance of mitochondrial translation defects as a causative factor is only starting to be recognised. We investigated the genetic basis for infantile onset recessive hypertrophic cardiomyopathy in two siblings.
Methods and results:
Analysis of respiratory chain enzymes revealed a combined deficiency of complexes I and IV in the heart and skeletal muscle. Exome sequencing uncovered a homozygous mutation (L156R) in MRPL44 of both siblings. MRPL44 encodes a protein in the large subunit of the mitochondrial ribosome and is suggested to locate in close proximity to the tunnel exit of the yeast mitochondrial ribosome. We found severely reduced MRPL44 levels in the patient's heart, skeletal muscle and fibroblasts suggesting that the missense mutation affected the protein stability. In patient fibroblasts, decreased MRPL44 affected assembly of the large ribosomal subunit and stability of 16S rRNA leading to complex IV deficiency. Despite this assembly defect, de novo mitochondrial translation was only mildly affected in fibroblasts suggesting that MRPL44 may have a function in the assembly/stability of nascent mitochondrial polypeptides exiting the ribosome. Retroviral expression of wild-type MRPL44 in patient fibroblasts rescued the large ribosome assembly defect and COX deficiency.
These findings indicate that mitochondrial ribosomal subunit defects can generate tissue-specific manifestations, such as cardiomyopathy.
Journal of Medical Genetics 01/2013; 50(3). DOI:10.1136/jmedgenet-2012-101375 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial encephalopathy, but the pathogenic role of the variant remained open. Clinical features, including postnatal onset, catastrophic epilepsy, lactic acidemia, early lethality and neuroimaging findings of the patients with FARS2 variants, resembled each other closely, and neuropathology was consistent with Alpers syndrome. Our structural analysis of mtPheRS predicted that p.I329T weakened ATP binding in the aminoacylation domain, and in vitro studies with recombinant mutant protein showed decreased affinity of this variant to ATP. Furthermore, p.D391V and p.Y144C were predicted to disrupt synthetase function by interrupting the rotation of the tRNA anticodon stem-binding domain from a closed to an open form. In vitro characterization indicated reduced affinity of p.D391V mutant protein to phenylalanine, whereas p.Y144C disrupted tRNA binding. The stability of p.I329T and p.D391V mutants in a refolding assay was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial aminoacyl-tRNA synthetase as a cause of mitochondrial disease.
Human Molecular Genetics 07/2012; 21(20):4521-9. DOI:10.1093/hmg/dds294 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To assess the long-term clinical course of carnitine palmitoyltransferase 1A (CPT1A) deficiency, caused by the c.1364A>C (p.K455T) mutation, and the carrier frequency of this mutation in Finland.
This was a long-term follow-up of patients in whom the common mutation was detected.
Between 1999 and 2010, 6 cases of CPT1A deficiency were diagnosed and treated with a high-carbohydrate, low-fat diet. The patients experienced their first symptoms during the first years of life, provoked by viral illness and/or fasting. The clinical features included hypoketotic hypoglycemia, hepatopathy, and loss of consciousness, ranging from transient unconsciousness to prolonged hyperlipidemic coma. Five cases carried a homozygous c.1364A>C (p.K455T) mutation, whereas 1 case had a compound c.1364A>C/c.1493A>C (p.Y498S) mutation. During dietary therapy, the patients had few transient decompensations. No carriers of mutation c.1364A>C were detected by minisequencing of 150 control samples.
Even though CPT1A deficiency may be life-threatening and lead to prolonged coma, the long-term prognosis is good. A genotype-phenotype correlation implies that the mutations detected are disease-causing. Despite Finland's location close to the Arctic polar region, the carrier frequency of the c.1364A>C mutation in Finland is far lower than that of the variants found in Alaskan, Canadian, and Greenland native populations.
The Journal of pediatrics 09/2011; 160(3):473-479.e1. DOI:10.1016/j.jpeds.2011.08.032 · 3.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in Pol γ represent a major cause of human mitochondrial diseases, especially those affecting the nervous system in adults and in children. Recessive mutations in Pol γ represent nearly half of those reported to date, and they are nearly uniformly distributed along the length of the POLG1 gene (Human DNA Polymerase gamma Mutation Database); the majority of them are linked to the most severe form of POLG syndrome, Alpers-Huttenlocher syndrome. In this report, we assess the structure-function relationships for recessive disease mutations by reviewing existing biochemical data on site-directed mutagenesis of the human, Drosophila and yeast Pol γs, and their homologs from the family A DNA polymerase group. We do so in the context of a molecular model of Pol γ in complex with primer-template DNA, which we have developed based upon the recently solved crystal structure of the apoenzyme form. We present evidence that recessive mutations cluster within five distinct functional modules in the catalytic core of Pol γ. Our results suggest that cluster prediction can be used as a diagnosis-supporting tool to evaluate the pathogenic role of new Pol γ variants.
Nucleic Acids Research 08/2011; 39(21):9072-84. DOI:10.1093/nar/gkr618 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infantile cardiomyopathies are devastating fatal disorders of the neonatal period or the first year of life. Mitochondrial dysfunction is a common cause of this group of diseases, but the underlying gene defects have been characterized in only a minority of cases, because tissue specificity of the manifestation hampers functional cloning and the heterogeneity of causative factors hinders collection of informative family materials. We sequenced the exome of a patient who died at the age of 10 months of hypertrophic mitochondrial cardiomyopathy with combined cardiac respiratory chain complex I and IV deficiency. Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS). Two siblings from another family, both of whom died perinatally of hypertrophic cardiomyopathy, had the same mutation, compound heterozygous with another missense mutation. Protein structure modeling of mtAlaRS suggested that one of the mutations affected a unique tRNA recognition site in the editing domain, leading to incorrect tRNA aminoacylation, whereas the second mutation severely disturbed the catalytic function, preventing tRNA aminoacylation. We show here that mutations in AARS2 cause perinatal or infantile cardiomyopathy with near-total combined mitochondrial respiratory chain deficiency in the heart. Our results indicate that exome sequencing is a powerful tool for identifying mutations in single patients and allows recognition of the genetic background in single-gene disorders of variable clinical manifestation and tissue-specific disease. Furthermore, we show that mitochondrial disorders extend to prenatal life and are an important cause of early infantile cardiac failure.
The American Journal of Human Genetics 05/2011; 88(5):635-42. DOI:10.1016/j.ajhg.2011.04.006 · 10.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial DNA polymerase γ (POLG1) mutations in children often manifest as Alpers syndrome, whereas in adults, a common manifestation is mitochondrial recessive ataxia syndrome (MIRAS) with severe epilepsy. Because some patients with MIRAS have presented with ataxia or epilepsy already in childhood, we searched for POLG1 mutations in neurologic manifestations in childhood.
We investigated POLG1 in 136 children, all clinically suspected to have mitochondrial disease, with one or more of the following: ataxia, axonal neuropathy, severe epilepsy without known epilepsy syndrome, epileptic encephalopathy, encephalohepatopathy, or neuropathologically verified Alpers syndrome.
Seven patients had POLG1 mutations, and all of them had severe encephalopathy with intractable epilepsy. Four patients had died after exposure to sodium valproate. Brain MRI showed parieto-occipital or thalamic hyperintense lesions, white matter abnormality, and atrophy. Muscle histology and mitochondrial biochemistry results were normal in all.
POLG1 analysis should belong to the first-line DNA diagnostic tests for children with an encephalitis-like presentation evolving into epileptic encephalopathy with liver involvement (Alpers syndrome), even if brain MRI and morphology, respiratory chain activities, and the amount of mitochondrial DNA in the skeletal muscle are normal. POLG1 analysis should precede valproate therapy in pediatric patients with a typical phenotype. However, POLG1 is not a common cause of isolated epilepsy or ataxia in childhood.
[Show abstract][Hide abstract] ABSTRACT: The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is a component of the respiratory chain of various bacteria that generates a redox-driven transmembrane electrochemical Na(+) potential. The Na(+)-NQR activity is known to be specifically inhibited by low concentrations of silver ions. Replacement of the conserved Cys377 residue with alanine in the NqrF subunit of Na(+)-NQR from Vibrio harveyi resulted in resistance of the enzyme to Ag(+) and to other heavy metal ions. Analysis of the catalytic activity also showed that the rate of electron input into the mutant Na(+)-NQR decreased by about 14-fold in comparison to the wild type enzyme, whereas all other properties of (NqrF)C377A Na(+)-NQR including its stability remained unaffected.
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial DNA polymerase, POLG, is the sole DNA polymerase found in animal mitochondria. In humans, POLGalpha W748S in cis with an E1143G mutation has been linked to a new type of recessive ataxia, MIRAS, which is the most common inherited ataxia in Finland. We investigated the biochemical phenotypes of the W748S amino acid change, using recombinant human POLG. We measured processive and non-processive DNA polymerase activity, DNA binding affinity, enzyme processivity, and subunit interaction with recombinant POLGbeta. In addition, we studied the effects of the W748S and E1143G mutations in primary human cell cultures using retroviral transduction. Here, we examined cell viability, mitochondrial DNA copy number, and products of mitochondrial translation. Our results indicate that the W748S mutant POLGalpha does not exhibit a clear biochemical phenotype, making it indistinguishable from wild type POLGalpha and as such, fail to replicate previously published results. Furthermore, results from the cell models were concurrent with the findings from patients, and support our biochemical findings.
[Show abstract][Hide abstract] ABSTRACT: Studies on the activity of Complex I from Escherichia coli in the presence of different metal cations revealed at least two high affinity metal-binding sites. Membrane-bound or isolated Complex I was activated by K(+) (apparent binding constant approximately 125 microM) and inhibited by La(3+) (IC(50)= 1 microM). K(+) and La(3+) do not occupy the same site. Possible localization of these metal-binding sites and their implication in catalysis are discussed.
[Show abstract][Hide abstract] ABSTRACT: Replacement of glutamate 95 for glutamine in the NADH- and FMN-binding NuoF subunit of E. coli Complex I decreased NADH oxidation activity 2.5-4.8 times depending on the used electron acceptor. The apparent K(m) for NADH was 5.2 and 10.4 microM for the mutant and wild type, respectively. Analysis of the inhibitory effect of NAD(+) on activity showed that the E95Q mutation caused a 2.4-fold decrease of K(i)(NAD+) in comparison to the wild type enzyme. ADP-ribose, which differs from NAD(+) by the absence of the positively charged nicotinamide moiety, is also a competitive inhibitor of NADH binding. The mutation caused a 7.5-fold decrease of K(i)(ADP-ribose) relative to wild type enzyme. Based on these findings we propose that the negative charge of Glu95 accelerates turnover of Complex I by electrostatic interaction with the negatively charged phosphate groups of the substrate nucleotide during operation, which facilitates release of the product NAD(+). The E95Q mutation was also found to cause a positive shift of the midpoint redox potential of the FMN, from -350 mV to -310 mV, which suggests that the negative charge of Glu95 is also involved in decreasing the midpoint potential of the primary electron acceptor of Complex I.
[Show abstract][Hide abstract] ABSTRACT: Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.
[Show abstract][Hide abstract] ABSTRACT: The redox properties of the cofactors of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli were studied by following the changes in electron paramagnetic resonance (EPR) and optical spectra upon electrochemical redox titration of the purified protein. At neutral pH, the FMN cofactor had a midpoint redox potential ( E m) approximately -350 mV ( n = 2). Binuclear FeS clusters were well-characterized: N1a was titrated with a single ( n = 1) transition, and E m = -235 mV. In contrast, the titration of N1b can only be fitted with the sum of at least two one-electron Nernstian curves with E m values of -245 and -320 mV. The tetranuclear clusters can also be separated into two groups, either having a single, n = 1, or more complex redox titration curves. The titration curves of the EPR bands attributed to the tetranuclear clusters N2 ( g = 2.045 and g = 1.895) and N6b ( g = 2.089 and g = 1.877) can be presented by the sum of at least two components, each with E m (app) approximately -200/-300 mV and -235/-315 mV, respectively. The titration of the signals at g = 1.956-1.947 (N3 or N7, E m = -315 mV), g = 2.022, and g = 1.932 (Nx, -365 mV) and the low temperature signal at g = 1.929 (N4 or N5, -330 mV) followed Nernstian n = 1 curves. The observed redox titration curves are discussed in terms of intrinsic electrostatic interactions between FeS centers in complex I. A model showing shifts of E m due to the electrostatic interaction between the centers is presented.
[Show abstract][Hide abstract] ABSTRACT: Electron transfer in complex I from Escherichia coli was investigated by an ultrafast freeze-quench approach. The reaction of complex I with NADH was stopped in the time domain from 90 mus to 8 ms and analyzed by electron paramagnetic resonance (EPR) spectroscopy at low temperatures. The data show that after binding of the first molecule of NADH, two electrons move via the FMN cofactor to the iron-sulfur (Fe/S) centers N1a and N2 with an apparent time constant of approximately 90 mus, implying that these two centers should have the highest redox potential in the enzyme. The rate of reduction of center N2 (the last center in the electron transfer sequence) is close to that predicted by electron transfer theory, which argues for the absence of coupled proton transfer or conformational changes during electron transfer from FMN to N2. After fast reduction of N1a and N2, we observe a slow, approximately 1-ms component of reduction of other Fe/S clusters. Because all elementary electron transfer rates between clusters are several orders of magnitude higher than this observed rate, we conclude that the millisecond component is limited by a single process corresponding to dissociation of the oxidized NAD(+) molecule from its binding site, where it prevents entry of the next NADH molecule. Despite the presence of approximately one ubiquinone per enzyme molecule, no transient semiquinone formation was observed, which has mechanistic implications, suggesting a high thermodynamic barrier for ubiquinone reduction to the semiquinone radical. Possible consequences of these findings for the proton translocation mechanism are discussed.
Proceedings of the National Academy of Sciences 04/2008; 105(10):3763-7. DOI:10.1073/pnas.0711249105 · 9.67 Impact Factor