Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a

We investigated the biochemical phenotype of the mtDNA T8993G point mutation in the ATPase 6 gene, associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families. All three carried >80% mutant genome in platelets and were manifesting clinically various degrees of the NARP phenotype. Coupled submitochondrial particles prepared from platelets capable of succinate-sustained ATP synthesis were studied using very sensitive and rapid luminometric and fluorescence methods. A sharp decrease (>95%) in the succinate-sustained ATP synthesis rate of the particles was found, but both the ATP hydrolysis rate and ATP-driven proton translocation (when the protons flow from the matrix to the cytosol) were minimally affected. The T8993G mutation changes the highly conserved residue Leu(156) to Arg in the ATPase 6 subunit (subunit a). This subunit, together with subunit c, is thought to cooperatively catalyze proton translocation and rotate, one with respect to the other, during the catalytic cycle of the F(1)F(0) complex. Our results suggest that the T8993G mutation induces a structural defect in human F(1)F(0)-ATPase that causes a severe impairment of ATP synthesis. This is possibly due to a defect in either the vectorial proton transport from the cytosol to the mitochondrial matrix or the coupling of proton flow through F(0) to ATP synthesis in F(1). Whatever mechanism is involved, this leads to impaired ATP synthesis. On the other hand, ATP hydrolysis that involves proton flow from the matrix to the cytosol is essentially unaffected.     

UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitoninpermeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.     

UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitoninpermeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.     

Partial uncoupling of the mitochondrial membrane by a heterozygous null mutation in the gene encoding the gamma- or delta-subunit of the yeast mitochondrial ATPase

Prior genetic studies indicated that the yeast mitochondrial ATP synthase can be assembled into enzyme complexes devoid of the gamma-, delta-, or epsilon-subunits (Lai-Zhang, J., Xiao, Y., and Mueller, D. M. (1999) EMBO J. 18, 58-64). These subunit-deficient complexes were postulated to uncouple the mitochondrial membrane thereby causing negative cellular phenotypes. This study provides biochemical and additional genetic data that support this hypothesis. The genetic data indicate that in a diploid cell, a heterozygous deletion mutation in the gene encoding the gamma- or delta-subunit of the ATPase is semidominant negative due to a decrease in the gene number from 2 to 1. However, the heterozygous atp2Delta mutation is epistatic to the heterozygous mutation in the gene encoding gamma or delta, suggesting that the semidominant negative effect is because of a gain of activity in the cells. Biochemical studies using mitochondria isolated from the yeast strains that are heterozygous for a mutation in gamma or delta indicate that the mitochondria are partially uncoupled. These results support the hypothesis that the negative phenotypes are caused by the formation of a gamma- or delta-less ATP synthase complex that is uncoupled.     

Reduction of the edited domain of the mitochondrial A6 gene for ATPase subunit 6 in Trypanosomatidae

The sequence of mitochondrial A6 (MURF4) was compared for several trypanosomatids in order to assess the reduction of the edited domain (ED). The association between the ED reduction and the phylogenetic position of a species proved to be less tight than believed earlier. Compared with digenetic species, monogenetic ones more often displayed ED reduction and had smaller ED.     

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F₁F_O-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F₁F_O complex to work in the reverse mode, i.e. F₁-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F₁, Atp9p-ring, unassembled α-F₁ subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.     

Cellular and functional analysis of four mutations located in the mitochondrial ATPase6 gene

The smallest rotary motor of living cells, F0F1‐ATP synthase, couples proton flow—generated by the OXPHOS system—from the intermembrane space back to the matrix with the conversion of ADP to ATP. While all mutations affecting the multisubunit complexes of the OXPHOS system probably impact on the cell's output of ATP, only mutations in complex V can be considered to affect this output directly. So far, most of the F0F1‐ATP synthase variations have been detected in the mitochondrial ATPase6 gene. In this study, the four most frequent mutations in the ATPase6 gene, namely L156R, L217R, L156P, and L217P, are studied for the first time together, both in primary cells and in cybrid clones. Arginine (“R”) mutations were associated with a much more severe phenotype than Proline (“P”) mutations, in terms of both biochemical activity and growth capacity. Also, a threshold effect in both “R” mutations appeared at 50% mutation load. Different mechanisms seemed to emerge for the two “R” mutations: the F1 seemed loosely bound to the membrane in the L156R mutant, whereas the L217R mutant induced low activity of complex V, possibly the result of a reduced rate of proton flow through the A6 channel. J. Cell. Biochem. 106: 878–886, 2009. © 2009 Wiley‐Liss, Inc.     

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

Background

Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein.

Results

In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody.

Conclusion

The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.     

Dimerization of a pathogenic human mitochondrial tRNA

Mutations of human mitochondrial transfer RNA (tRNA) are implicated in a variety of multisystemic diseases. The most prevalent pathogenic mitochondrial mutation is the A3243G substitution within the gene for tRNA(Leu(UUR)). Here we describe the pronounced structural change promoted by this mutation. The A3243G mutation induces the formation of a tRNA dimer that strongly self-associates under physiological conditions. The dimerization interface in the mutant tRNA is a self-complementary hexanucleotide in the D-stem, a particularly weak structural element within tRNA(Leu(UUR)). Aminoacylation of the A3243G mutant is significantly attenuated, and mutational studies indicate that dimerization is partially responsible for the observed loss of function. The disruption of a conserved tertiary structural contact also contributes to the functional defect. The pathogenic mutation is proposed to interfere with the cellular function of human mitochondrial tRNA(Leu(UUR)) by destabilizing the native structure and facilitating the formation of a dimeric complex with low biological activity.     

A single amino acid change in subunit 6 of the yeast mitochondrial ATPase suppresses a null mutation in ATP10

In an earlier study, the ATP10 gene of Saccharomyces cerevisiae was shown to code for an inner membrane protein required for assembly of the F(0) sector of the mitochondrial ATPase complex (Ackerman, S., and Tzagoloff, A. (1990) J. Biol. Chem. 265, 9952-9959). To gain additional insights into the function of Atp10p, we have analyzed a revertant of an atp10 null mutant that displays partial recovery of oligomycin-sensitive ATPase and of respiratory competence. The suppressor mutation in the revertant has been mapped to the OLI2 locus in mitochondrial DNA and shown to be a single base change in the C-terminal coding region of the gene. The mutation results in the substitution of a valine for an alanine at residue 249 of subunit 6 of the ATPase. The ability of the subunit 6 mutation to compensate for the absence of Atp10p implies a functional interaction between the two proteins. Such an interaction is consistent with evidence indicating that the C-terminal region with the site of the mutation and the extramembrane domain of Atp10p are both on the matrix side of the inner membrane. Subunit 6 has been purified from the parental wild type strain, from the atp10 null mutant, and from the revertant. The N-terminal sequences of the three proteins indicated that they all start at Ser(11), the normal processing site of the subunit 6 precursor. Mass spectral analysis of the wild type and mutants subunit 6 failed to reveal any substantive difference of the wild type and mutant proteins when the mass of the latter was corrected for Ala --> Val mutation. These data argue against a role of Atp10p in post-translational modification of subunit 6. Although post-translational modification of another ATPase subunit interacting with subunit 6 cannot be excluded, a more likely function for Atp10p is that it acts as a subunit 6 chaperone during F(0) assembly.     

Hereditary spastic paraplegia-like disorder due to a mitochondrial ATP6 gene point mutation

Hereditary spastic paraplegia refers to a genetically heterogeneous syndrome. We identified five members of a family suffering from a late-onset spastic paraplegia-like disorder, carrying the homoplasmic m.9176 T > C mutation in the mitochondrial ATP6 gene. The clinical severity of the disease observed in the family was correlated with the biochemical and assembly defects of the ATP synthase. The m.9176 T > C mutation has been previously associated to Leigh syndrome or familial bilateral striatal necrosis. Other factors such as modifying genes may be involved in the phenotypic expression of the disease. The family belongs to the mitochondrial haplogroup J, previously shown to play a role in modulating the phenotype of mitochondrial diseases and be associated with longevity. Moreover other nuclear modifying genes or environmental factors may contribute to the disease phenotype. This finding extends the genetic heterogeneity of the hereditary spastic paraplegia together with the clinical spectrum of mutations of the ATP6 gene.     

Decreased CCA-addition in human mitochondrial tRNAs bearing a pathogenic A4317G or A10044G mutation

Pathogenic point mutations in mitochondrial tRNA genes are known to cause a variety of human mitochondrial diseases. Reports have associated an A4317G mutation in the mitochondrial tRNA(Ile) gene with fatal infantile cardiomyopathy and an A10044G mutation in the mitochondrial tRNA(Gly) gene with sudden infant death syndrome. Here we demonstrate that both mutations inhibit in vitro CCA-addition to the respective tRNA by the human mitochondrial CCA-adding enzyme. Structures of these two mutant tRNAs were examined by nuclease probing. In the case of the A4317G tRNA(Ile) mutant, structural rearrangement of the T-arm region, conferring an aberrantly stable T-arm structure and an increased T(m) value, was clearly observed. In the case of the A10044G tRNA(Gly) mutant, high nuclease sensitivity in both the T- and D-loops suggested a weakened interaction between the loops. These are the first reported instances of inefficient CCA-addition being one of the apparent molecular pathogeneses caused by pathogenic point mutations in human mitochondrial tRNA genes.     

Oligomycin A induces autophagy in the IPLB-LdFB insect cell line

Functional and morphological modifications in the IPLB-LdFB insect cell line were examined following a short treatment with a reversible inhibitor of mitochondrial ATP synthase, oligomycin A, and subsequent incubation for various times in oligomycin-A-free medium. Oncosis, apoptosis and autophagy at variable percentages were observed under the various experimental conditions. Together with oncotic and apoptotic pathways that lead directly to cell death, the insect cells responded to ATP depletion with autophagy. Our results revealed that, in most cases, autophagy failed to restore cellular homeostasis, probably because of a massive sequestration of mitochondria in autophagic vacuoles. This critical event was a “point of no return” and ultimately resulted in cell necrosis. However, cells with a misshapen body and nucleus resembling “resistant forms” were observed at the end of the experiments. Our findings indicate that oligomycin-A-induced autophagy can promote cell protection or cell destruction and is an open-ended process that can lead to survival or death depending on a combination of concomitant factors.This work was supported by MIUR (Italy) grants to M.deE. and E.O. and by the Centro Grandi Attrezzature (University of Insubria, Varese, Italy).Gianluca Tettamanti and Davide Malagoli contributed equally to this work.