Mitochondrial Protein Extracts Research Articles

Divergent Mitochondrial Antioxidant Activities and Lung Alveolar Architecture in the Lungs of Rats and Mice at High Altitude.

Compared with mice, adult rats living at 3,600 m above sea level (SL—La Paz, Bolivia) have high hematocrit, signs of pulmonary hypertension, and low lung volume with reduced alveolar surface area. This phenotype is associated with chronic mountain sickness in humans living at high altitude (HA). We tested the hypothesis that this phenotype is associated with impaired gas exchange and oxidative stress in the lungs. We used rats and mice (3 months old) living at HA (La Paz) and SL (Quebec City, Canada) to measure arterial oxygen saturation under graded levels of hypoxia (by pulse oximetry), the alveolar surface area in lung slices and the activity of pro- (NADPH and xanthine oxidases—NOX and XO) and anti- (superoxide dismutase, and glutathione peroxidase—SOD and GPx) oxidant enzymes in cytosolic and mitochondrial lung protein extracts. HA rats have a lower arterial oxygen saturation and reduced alveolar surface area compared to HA mice and SL rats. Enzymatic activities (NOX, XO, SOD, and GPx) in the cytosol were similar between HA and SL animals, but SOD and GPx activities in the mitochondria were 2–3 times higher in HA vs. SL rats, and only marginally higher in HA mice vs. SL mice. Furthermore, the maximum activity of cytochrome oxidase-c (COX) measured in mitochondrial lung extracts was also 2 times higher in HA rats compared with SL rats, while there was only a small increase in HA mice vs. SL mice. Interestingly, compared with SL controls, alterations in lung morphology are not observed for young rats at HA (15 days after birth), and enzymatic activities are only slightly altered. These results suggest that rats living at HA have a gradual reduction of their alveolar surface area beyond the postnatal period. We can speculate that the elevation of SOD, GPx, and COX activities in the lung mitochondria are not sufficient to compensate for oxidative stress, leading to damage of the lung tissue in rats.

DNA Polymerase Beta Participates in Mitochondrial DNA Repair.

We have detected DNA polymerase beta (Polβ), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polβ in the mitochondria. Using Polβ fragments, mitochondrion-specific protein partners were identified, with the interactors functioning mainly in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1, and TFAM, all of which are mitochondrion-specific DNA effectors and are known to function in the nucleoid. Polβ directly interacted functionally with the mitochondrial helicase TWINKLE. Human kidney cells with Polβ knockout (KO) had higher endogenous mitochondrial DNA (mtDNA) damage. Mitochondrial extracts derived from heterozygous Polβ mouse tissue and KO cells had lower nucleotide incorporation activity. Mouse-derived Polβ null fibroblasts had severely affected metabolic parameters. Indeed, gene knockout of Polβ caused mitochondrial dysfunction, including reduced membrane potential and mitochondrial content. We show that Polβ is a mitochondrial polymerase involved in mtDNA maintenance and is required for mitochondrial homeostasis.

Cyclophilin D over-expression increases mitochondrial complex III activity and accelerates supercomplex formation

Cyclophilin D (CyPD), a mitochondrial matrix protein, has been widely studied for its role in mitochondrial-mediated cell death. Unexpectedly, we previously discovered that overexpression of CyPD in a stable cell line, increased mitochondrial membrane potentials and enhanced cell survival under conditions of oxidative stress. Here, we investigated the underlying mechanisms responsible for these findings. Spectrophotometric measurements in isolated mitochondria revealed that overexpression of CyPD in HEK293 cells increased respiratory chain activity, but only for Complex III (CIII). Acute treatment of mitochondria with the immumosupressant cyclosporine A did not affect CIII activity. Expression levels of the CIII subunits cytochrome b and Rieske-FeS were elevated in HEK293 cells overexpressing CyPD. However, CIII activity was still significantly higher compared to control mitochondria, even when normalized by protein expression. Blue native gel electrophoresis and Western blot assays revealed a molecular interaction of CyPD with CIII and increased levels of supercomplexes in mitochondrial protein extracts. Radiolabeled protein synthesis in mitochondria showed that CIII assembly and formation of supercomplexes containing CIII were significantly faster when CyPD was overexpressed. Taken together, these data indicate that CyPD regulates mitochondrial metabolism, and likely cell survival, by promoting more efficient electrons flow through the respiratory chain via increased supercomplex formation.

Structure and Biophysical Characterization of the S-Adenosylmethionine-dependent O-Methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina

Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O-Methylation is performed by O-methyltransferases, members of the S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remain unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor), and PaMTH1-S-adenosyl homocysteine (by-product) co-complexes refined to 2.0, 1.9, and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N termini. Each monomer adopts the Rossmann fold typical for many SAM-binding methyltransferases. Structural comparisons between different O-methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket, indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry, and site-directed active site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner.

A quantitative assay for reductive metabolism of a pesticide in fish using electrochemistry coupled with liquid chromatography tandem mass spectrometry.

This is the first study to use electrochemistry to generate a nitro reduction metabolite as a standard for a liquid chromatography-mass spectrometry-based quantitative assay. This approach is further used to quantify 3-trifluoromethyl-4-nitrophenol (TFM) reductive metabolism. TFM is a widely used pesticide for the population control of sea lamprey (Petromyzon marinus), an invasive species of the Laurentian Great Lakes. Three animal models, sea lamprey, lake sturgeon (Acipenser fulvescens), and rainbow trout (Oncorhynchus mykiss), were selected to evaluate TFM reductive metabolism because they have been known to show differential susceptibilities to TFM toxicity. Amino-TFM (aTFM; 3-trifluoromethyl-4-aminophenol) was the only reductive metabolite identified through liquid chromatography-high-resolution mass spectrometry screening of liver extracts incubated with TFM and was targeted for electrochemical synthesis. After synthesis and purification, aTFM was used to develop a quantitative assay of the reductive metabolism of TFM through liquid chromatography and tandem mass spectrometry. The concentrations of aTFM were measured from TFM-treated cellular fractions, including cytosolic, nuclear, membrane, and mitochondrial protein extracts. Sea lamprey extracts produced the highest concentrations (500 ng/mL) of aTFM. In addition, sea lamprey and sturgeon cytosolic extracts showed concentrations of aTFM substantially higher than those of rainbow trout. However, other fractions of lake sturgeon extracts tend to show aTFM concentrations similar to those of rainbow trout but not with sea lamprey. These data suggest that the level of reductive metabolism of TFM may be associated with the sensitivities of the animals to this particular pesticide.

Activation of Bax in three models of retinitis pigmentosa.

The process of photoreceptor cell death in retinitis pigmentosa is still not well characterized, and identification of common mechanisms will be instrumental for development of therapeutic strategies. Here we investigated activation of Bax in rd1, P23H transgenic, and Rho knockout retinas. Bax activation was evaluated by immunofluorescence using anti-activated Bax-specific antibodies and by Western blotting on mitochondrial protein extracts. Knockdown of cathepsin D, calpain 1, and calpain 2 was achieved by short hairpin RNA (shRNA) delivery in rd1 mutant photoreceptors cells differentiated from retinal neurospheres. The mechanism of Bax activation through calpains was evaluated in vivo by intravitreal injection of calpastatin. We defined activation and mitochondrial localization of Bax as well as activation of calpains and cathepsin D in the three models of retinitis pigmentosa. Taking advantage of an in vitro culture system for rd1 mutant photoreceptors, we unraveled the mechanism of Bax activation. We demonstrated that calpain 1 and cathepsin D contributed to activation of Bax and to apoptosis-inducing factor (Aif) nuclear translocation. In vivo interference with calpain activity blocks Bax activation in the rd1 and Rho knockout retinas and reduces activation in the P23H transgenic retina. Activation of Bax was observed in all three models of retinitis pigmentosa and leads to neurodamage by localization at the mitochondrion. Our data suggest that Bax can be envisaged as one of the promising target molecules for restraining photoreceptor degeneration.

Systematic Exploration of Thioredoxin Target Proteins in Plant Mitochondria

The thioredoxin (Trx) system is known to play a pivotal role in cellular redox regulation, but its target proteins in plant mitochondria remain largely uncharacterized. In this study, we systemically screened Trx target candidates in plant mitochondria. Mitochondrial protein extracts were prepared from Arabidopsis shoots, spinach leaves and potato tubers, and then subfractionated into soluble matrix and insoluble membrane fractions. Protein extracts were loaded onto an affinity column immobilizing Arabidopsis mitochondria-localized o-type Trx mutant protein, in which one of two internal cysteines at the active site was substituted by serine. Proteins forming mixed-disulfide intermediates with the mutated Trx were identified by proteomic approaches. This procedure allowed the determination of 101 Trx target candidate proteins involved in a broad spectrum of mitochondrial processes. Furthermore, biochemical assay revealed that one of the potential Trx target proteins, alternative oxidase, is actually redox regulated by Trx. This study provides insights into the regulatory mechanism of diverse functions in mitochondrial biology that are mediated through the Trx system.

A three‐way proteomics strategy allows differential analysis of yeast mitochondrial membrane protein complexes under anaerobic and aerobic conditions

To investigate the effect of anaerobiosis on the Saccharomyces cerevisiae mitochondrial proteome and the formation of respiratory chain and other protein complexes, we analyzed mitochondrial protein extracts that were enriched from lysates of aerobic and anaerobic steady-state chemostat cultures. We chose an innovative approach in which native mitochondrial membrane protein complexes were separated by 1-D blue native PAGE, which was combined with quantitative analysis of each complex subunit using stable isotope labeling. LC-FT(ICR)-MS/MS analysis was applied to identify and quantify the mitochondrial proteins. In addition, to establish if changes in mitochondrial complex composition occurred under anaerobiosis, we investigated the 1-D blue native PAGE protein migration patterns by Pearson correlation analysis. Surprisingly, we discovered that under anaerobic conditions, where the yeast respiratory chain is not active, the respiratory chain supercomplexes, such as complex V dimer, complex (III)(2)(IV)(2) and complex (III)(2)(IV) were still present, although at reduced levels. Pearson correlation analysis showed that the composition of the mitochondrial complexes was unchanged under aerobic or anaerobic conditions, with the exception of complex II. In addition, this latter approach allowed screening for possible novel complex interaction partners, since for example protein Aim38p, with a yet unknown function, was identified as a possible component of respiratory chain complex IV.

Rat mitochondrial manganese superoxide dismutase: Amino acid positions involved in covalent modifications, activity, and heat stability

The role of three amino acid residues (Q143, Y34, S82) of rat mitochondrial superoxide dismutase (ratSOD2) in the enzymatic activity, thermostability, and post-translational modification of the enzyme was investigated through site-directed mutagenesis studies. Six recombinant forms of the enzyme were produced, carrying the Q143 or H143 residue with or without the Y34F or S82A replacement. All proteins bound manganese as active cofactor and were organized as homotetramers. The greatest effect on the activity (sixfold reduction) was observed in ratSOD2 forms containing the H143 variant, whereas Y34F and S82A substitutions moderately reduced the enzymatic activity compared to the Q143 form. Heat inactivation studies showed the high thermo-tolerance of ratSOD2 and allowed an evaluation of the related activation parameters of the heat inactivation process. Compared to Q143, the H143 variant was significantly less heat stable and displayed moderately lower enthalpic and entropic factors; the Y34F substitution caused a moderate reduction of heat stability, whereas the S82A replacement slightly improved the thermo-tolerance of the Q143 variant; both substitutions significantly increased enthalpic and entropic factors of heat inactivation, the greatest effect being observed with S82A substitution. All recombinant forms of ratSOD2 were glutathionylated in Escherichia coli, a feature pointing to the high reactivity of ratSOD2 toward glutathione. Moreover, the S82 position of the enzyme was phosphorylated in an in vitro system containing human mitochondrial protein extracts as source of protein kinases. These data highlight the role played by some residues in ratSOD2 and suggest a fine regulation of the enzyme occurring in vivo.

Oxidative DNA damage and its repair in rat spleen following subchronic exposure to aniline

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Splenotoxicity of aniline is associated with iron overload and generation of reactive oxygen species (ROS) which can cause oxidative damage to DNA, proteins and lipids (oxidative stress). 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the most abundant oxidative DNA lesions resulting from ROS, and 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase/lyase enzyme, plays a key role in the removal of 8-OHdG adducts. This study focused on examining DNA damage (8-OHdG) and repair (OGG1) in the spleen in an experimental condition preceding a tumorigenic response. To achieve that, male Sprague–Dawley rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. Aniline treatment led to a significant increase in splenic oxidative DNA damage, manifested as a 2.8-fold increase in 8-OHdG levels. DNA repair activity, measured as OGG1 base excision repair (BER) activity, increased by ∼ 1.3 fold in the nuclear protein extracts (NE) and ∼ 1.2 fold in the mitochondrial protein extracts (ME) of spleens from aniline-treated rats as compared to the controls. Real-time PCR analysis for OGG1 mRNA expression in the spleen revealed a 2-fold increase in expression in aniline-treated rats than the controls. Likewise, OGG1 protein expression in the NEs of spleens from aniline-treated rats was ∼ 1.5 fold higher, whereas in the MEs it was ∼ 1.3 fold higher than the controls. Aniline treatment also led to stronger immunostaining for both 8-OHdG and OGG1 in the spleens, confined to the red pulp areas. It is thus evident from our studies that aniline-induced oxidative stress is associated with increased oxidative DNA damage. The BER pathway was also activated, but not enough to prevent the accumulation of oxidative DNA damage (8-OHdG). Accumulation of mutagenic oxidative DNA lesions in the spleen following exposure to aniline could play a critical role in the tumorigenic process.

Mitochondrial Transcription Factor B2 Is Essential for Metabolic Function in Drosophila melanogaster Development

Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development.

The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA

The mammalian mitochondrial transcription termination factor mTERF binds with high affinity to a site within the tRNALeu(UUR) gene and regulates the amount of read through transcription from the ribosomal DNA into the remaining genes of the major coding strand of mitochondrial DNA (mtDNA). Electrophoretic mobility shift assays (EMSA) and SELEX, using mitochondrial protein extracts from cells induced to overexpress mTERF, revealed novel, weaker mTERF-binding sites, clustered in several regions of mtDNA, notably in the major non-coding region (NCR). Such binding in vivo was supported by mtDNA immunoprecipitation. Two-dimensional neutral agarose gel electrophoresis (2DNAGE) and 5′ end mapping by ligation-mediated PCR (LM-PCR) identified the region of the canonical mTERF-binding site as a replication pause site. The strength of pausing was modulated by the expression level of mTERF. mTERF overexpression also affected replication pausing in other regions of the genome in which mTERF binding was found. These results indicate a role for TERF in mtDNA replication, in addition to its role in transcription. We suggest that mTERF could provide a system for coordinating the passage of replication and transcription complexes, analogous with replication pause-region binding proteins in other systems, whose main role is to safeguard the integrity of the genome whilst facilitating its efficient expression.

In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno‐associated virus (rAAV) serotypes 2 and 8

Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B(12), adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). The human ATR cDNA was cloned into a recombinant adeno-associated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/Bl6 mice at a dose of 1 x 10(10) and 1 x 10(11) particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken beta-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments.

A novel interaction between DNA ligase III and DNA polymerase γ plays an essential role in mitochondrial DNA stability

The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.

115. In Vivo Expression of Human Cob(I)alamin Adenosyltransferase Using rAAV Serotypes 1,2 and 8, an Approach to Gene Therapy for Methylmalonic Aciduria (MMA)

Top of pageAbstract Methylmalonic aciduria (MMA) is an autosomal recessive disease with an incidence of approximately 1 in 50,000 people in the United States. Symptoms include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000 fold greater then normal. Currently, eight different complementation groups (cblA-H) for MMA have been described. CblB MMA is caused by a deficiency in the enzyme Cob(I)alamin adenosyltransferase (ATR). ATR functions within the mitochondria matrix in the final conversion of inactive vitamin B12 to coenzyme B12, adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). AdoCbl and MCM catalyze methylmalonyl-CoA to succinyl-CoA, which is further metabolized in the TCA cycle. The human ATR cDNA was cloned into an rAAV vector containing the hybrid cytomegalovirus (CMV) enhancer with the chicken beta-actin promoter and AAV2-inverted terminal repeats and pseudotyped in AAV1, 2 or 8 capsids. AAV serotypes 2 and 8 were delivered by portal vein injection to six week old female C57/Bl6 mice at a low dose of 1|[times]|1010 particles and a high dose of 1|[times]|1011 particles. Eight-weeks post injection, these animals were sacrificed and their livers harvested. RNA, genomic DNA and protein were then extracted from these tissues and used for reverse transcriptase PCR, real time PCR, western blotting and ATR enzyme assays. Portions of the liver were also reserved for immunostaining. AAV1 and 2 were delivered intramuscularly to the tibialis anterior of C57/Bl6 at a low dose of 1|[times]|1010 and a high dose of 1|[times]|1011 particles. These animals were sacrificed at ten weeks and the injected muscles were harvested. RT-PCR was used to confirm transcript levels, and immunostaining was performed for tissue biodistribution. RESULTS: Using primer pairs specific to the CMV enhancer/chicken beta-actin promoter of rAAV vectors, genome copies/cell in the liver were found to 0.03, 2.03 and 0.10 for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Using primer pairs specific for the vector genome, a substantial increase of message was seen with AAV8 high dose over AAV2 dose. Similar mRNA expression was seen between the AAV8 low dose and AAV2 high dose animal tissues. Western blotting performed on mitochondrial protein extracts demonstrated the presence of pre-ATR and post-translocated protein in all three dose groups. Levels were comparable to control levels in the AAV8 low dose and AAV2 high dose animals. Levels three to five fold higher than control levels were observed in AAV8 high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the AAV8 high dose animals, compared to 9% and 5% transduction in AAV2 high dose and AAV8 low dose animals, respectively. In muscle, an increase of rAAV ATR message was seen in AAV1 high dose animal muscles as compared to AAV2 high dose animals.

Nuclear Control of Cloverleaf Structure of Human Mitochondrial tRNA Lys

The evolutionary loss in eukaryotic cells of mitochondrial (mt) tRNA genes and of tRNA structural information in the surviving genes has led to the appearance of mt-tRNAs with highly unusual structural features. One such mt-tRNA is the human mt-tRNA Lys, which relies on post-transcriptional base modification to achieve correct three-dimensional structure. It has been shown that the in vitro transcript of human mt-tRNA Lys adopts a particular, non-cloverleaf structure when devoid of modified bases, while the native, fully modified tRNA shows the expected cloverleaf structure. Furthermore, a methyl group at position A9- N 1, introduced chemically in an otherwise unmodified mt-tRNA Lys transcript, was found to induce a stable cloverleaf conformation, raising the question of how the specific methyltransferase recognizes the unmodified transcript. In order to shed light on this unusual case of tRNA maturation, the tRNA modification enzymes contained in protein extracts from either highly purified HeLa cell mitochondria or HeLa cell cytosol were first identified and compared, and then used to analyze the mt-tRNA Lys. An initial screening for modification activities, using as substrates unmodified in vitro transcripts of tRNA genes with well characterized structures, namely yeast cytosolic tRNA Phe, human cytosolic tRNA Lys 3, and human mt-tRNA Ile, revealed the presence of nine and 11 modification activities in the mitochondrial and cytosolic protein extracts, respectively, the mitochondrial extract including a tRNA (adenine-9, N 1)-methyltransferase activity. The comparison of the level and kinetics of A9- N 1 methylation and other secondary modifications in the unmodified, misfolded mt-tRNA Lys and in a cloverleaf-shaped structural mutant, engineered to adopt the tRNA Lys cloverleaf structure without post-transcriptional modifications, suggested strongly that the methylation of A9- N 1 in tRNA Lys proceeds via a cloverleaf-shaped intermediate. Therefore, it is proposed that this intermediate is present in the in vitro transcript as part of a dynamic equilibrium, and that the mitochondrial protein extract contains an activity that stabilizes, by secondary modification, such a transient cloverleaf-shaped intermediate. Thus, countering the evolutionary loss of structural information in mt-tRNA genes, the mt-tRNA structure is maintained by a modification enzyme encoded in nuclear DNA.

Age-dependent decline of DNA repair activity for oxidative lesions in rat brain mitochondria.

Endogenous oxidative damage to brain mitochondrial DNA and mitochondrial dysfunction are contributing factors in aging and in the pathogenesis of a number of neurodegenerative diseases. In this study, we characterized the regulation of base-excision-repair (BER) activity, the predominant repair mechanism for oxidative DNA lesions, in brain mitochondria as the function of age. Mitochondrial protein extracts were prepared from rat cerebral cortices at the ages of embryonic day 17 (E17) or postnatal 1-, 2-, and 3-weeks, or 5- and 30-months. The total BER activity and the activity of essential BER enzymes were examined in mitochondria using in vitro DNA repair assay employing specific repair substrates. Mitochondrial BER activity showed marked age-dependent declines in the brain. The levels of overall BER activity were highest at E17, gradually decreased thereafter, and reached to the lowest at the age of 30-month ( approximately 80% reduction). The decline of overall BER activity with age was attributed to the decreased expression of repair enzymes such as 8-OHdG glycosylase and DNA polymerase-gamma and, consequently, the reduced activity at the steps of lesion-base incision, DNA repair synthesis and DNA ligation in the BER pathway. These results strongly suggest that the decline in BER activity may be an important mechanism contributing to the age-dependent accumulation of oxidative DNA lesions in brain mitochondria.

Mitochondrial DNA ligase function in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Deltadnl4). The Escherichia coli ECO:RI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant ECO:RI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Deltadnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.

Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity.

The human DNA ligase III gene encodes both nuclear and mitochondrial proteins. Abundant evidence supports the conclusion that the nuclear DNA ligase III protein plays an essential role in both base excision repair and homologous recombination. However, the role of DNA ligase III protein in mitochondrial genome dynamics has been obscure. Human tumor-derived HT1080 cells were transfected with an antisense DNA ligase III expression vector and clones with diminished levels of DNA ligase III activity identified. Mitochondrial protein extracts prepared from these clones had decreased levels of DNA ligase III relative to extracts from cells transfected with a control vector. Analysis of these clones revealed that the DNA ligase III antisense mRNA-expressing cells had reduced mtDNA content compared to control cells. In addition, the residual mtDNA present in these cells had numerous single-strand nicks that were not detected in mtDNA from control cells. Cells expressing antisense ligase III also had diminished capacity to restore their mtDNA to pre-irradiation levels following exposure to gamma-irradiation. An antisense-mediated reduction in cellular DNA ligase IV had no effect on the copy number or integrity of mtDNA. This observation, coupled with other evidence, suggests that DNA ligase IV is not present in the mitochondria and does not play a role in maintaining mtDNA integrity. We conclude that DNA ligase III is essential for the proper maintenance of mtDNA in cultured mammalian somatic cells.

Alterations in mitochondria and mtTFA in response to LPS-induced differentiation of B-cells

Stimulation of immune cells results in altered cell function and metabolism, which must be recognized by and coordinated with energy production from mitochondria. Mitochondria contain their own DNA genome encoding 13 polypeptides that combine with nuclear-derived subunits to create functional enzyme complexes of the electron transport chain. Therefore, coordination of mitochondrial and nuclear transcription is necessary to achieve a sustained elevation in mitochondrial ATP production. Pre-B-lymphocytes stimulated with lipopolysaccharide exhibit increased activity levels of the mitochondrial enzymes, succinate dehydrogenase and cytochrome c oxidase. Immunoblot analyses of purified mitochondria indicate an increase in the mitochondrial transcription factor (mtTFA) levels in mitochondria induced by cell stimulation. This increase is consistent with increased mtTFA production in the cytoplasm. In addition, mitochondrial protein extracts indicate an increase in protein binding to a mtTFA–DNA binding site from the mitochondrial genome, subsequent to cell stimulation. These results indicate that mitochondrial activity changes during B-lymphocyte stimulation, and mtTFA may contribute to the coordination of respiration with cellular energy demand.