Taurine as a constituent of mitochondrial tRNAs: new insights into the functions of taurine and human mitochondrial diseases

Taurine (2-aminoethanesulphonic acid), a naturally occurring, sulfur-containing amino acid, is found at high concentrations in mammalian plasma and tissues. Although taurine is involved in a variety of processes in humans, it has never been found as a component of a protein or a nucleic acid, and its precise biochemical functions are not fully understood. Here, we report the identification of two novel taurine-containing modified uridines (5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine) in human and bovine mitochondrial tRNAs. Our work further revealed that these nucleosides are synthesized by the direct incorporation of taurine supplied to the medium. This is the first reported evidence that taurine is a constituent of biological macromolecules, unveiling the prospect of obtaining new insights into the functions and subcellular localization of this abundant amino acid. Since modification of these taurine-containing uridines has been found to be lacking in mutant mitochondrial tRNAs for Leu(UUR) and Lys from pathogenic cells of the mitochondrial encephalomyopathies MELAS and MERRF, respectively, our findings will considerably deepen our understanding of the molecular pathogenesis of mitochondrial encephalomyopathic diseases.     

Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases

Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNA(Leu(UUR)) with a MELAS A3243G mutation and mt tRNA(Lys) with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [taum(5)(s(2))U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.     

Diseases caused by mutations in mitochondrial DNA

Mitochondrial diseases associated with mutations within mitochondrial genome are a subgroup of metabolic disorders since their common consequence is reduced metabolic efficiency caused by impaired oxidative phophorylation and shortage of ATP. Although the vast majority of mitochondrial proteins (approximately 1500) is encoded by nuclear genome, mtDNA encodes 11 subunits of respiratory chain complexes, 2 subunits of ATP synthase, 22 tRNAs and 2 rRNAs. Up to now, more than 250 pathogenic mutations have been described within mtDNA. The most common are point mutations in genes encoding mitochondrial tRNAs such as 3243A-->G and 8344T-->G that cause, respectively, MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) or MIDD (maternally-inherited diabetes and deafness) and MERRF (myoclonic epilepsy with ragged red fibres) syndromes. There have been also found mutations in genes encoding subunits of ATP synthase such as 8993T-->G substitution associated with NARP (neuropathy, ataxia and retinitis pigmentosa) syndrome. It is worth to note that mitochondrial dysfunction can also be caused by mutations within nuclear genes coding for mitochondrial proteins.     

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.     

Transfer RNA genes in the cap-oxil region of yeast mitochondrial DNA.

A cytoplasmic "petite" (rho-) clone of Saccharomyces cerevisiae has been isolated and found through DNA sequencing to contain the genes for cysteine, histidine, leucine, glutamine, lysine, arginine, and glycine tRNAs. This clone, designated DS502, has a tandemly repeated 3.5 kb segment of the wild type genome from 0.7 to 5.6 units. All the tRNA genes are transcribed from the same strand of DNA in the direction cap to oxil. The mitochondrial DNA segment of DS502 fills a sequence gap that existed between the histidine and lysine tRNAs. The new sequence data has made it possible to assign accurate map positions to all the tRNA genes in the cap-oxil span of the yeast mitochondrial genome. A detailed restriction map of the region from 0 to 17 map units along with the locations of 16 tRNA genes have been determined. The secondary structures of the leucine and glutamine tRNAs have been deduced from their gene sequences. The leucine tRNA exhibits 64% sequence homology to an E. coli leucine tRNA.     

MELAS和MERRF综合征相关mtDNA突变位点检测集成芯片的建立

摘要: 文中建立了一种新型的寡核苷酸芯片, 用于线粒体脑肌病伴高乳酸血症和卒中样发作(Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes, MELAS)和肌阵挛性癫痫伴发不规整红纤维(Myoclonic epilepsy with ragged red fibers, MERRF)线粒体DNA所有已知突变位点的集成检测。将31对allele位点特异性的寡核苷酸探针包被在醛基修饰的载玻片表面, 以多重不对称PCR方法制备Cy5荧光标记靶基因。利用此芯片对5例MELAS患者、5例MERRF患者及20例健康对照进行筛查, 结果发现, MELAS患者均为MT-T1基因A3243G突变; 在MERRF患者组, MT-TK基因A8344G突变4例, T8356C突变1例; 健康对照组均未发现31种相关mtDNA突变。芯片检测与DNA测序结果完全一致。结果表明, 这种寡核苷酸芯片可以对MELAS和MERRF综合征已知突变位点进行同步快速检测, 具有较高的灵敏度和特异性。这一模式的基因芯片经过适当改装后也可用于其他人类线粒体疾病的基因诊断。     

Diagnosis of defects in oxidative muscle metabolism by non-invasive tissue oximetry

The dynamics of oxygen delivery and utilization are examined in a variety of mitochondrial disorders during rest, exercise and post exercise. We used a non-invasive optical technique to measure the oxygen consumption in the exercising limb in normal subjects and 5 patients with cytochrome c oxidase deficiency. We also examined 6 patients with MELAS and MERRF syndrome. We measured near-infrared spectra of hemoglobin in the gastrocnemius muscle during treadmill exercise. Normal subjects demonstrated a sustained deoxygenation during exercise, indicating an efficient utilization of delivered oxygen. Patients with cytochrome c oxidase deficiency demonstrated consistent oxygenation during exercise indicating an under utilization of delivered oxygen. Patients with MELAS and MERRF syndrome showed similar under utilization of oxygen during exercise. Non-invasive tissue oximetry during exercise demonstrates specific abnormalities in a variety of mitochondrial disorders, indicating abnormal oxygen utilization, and will be a useful addition to the clinical investigation of such disorders. (Mol Cell Biochem 174: 7–10, 1997)     

Selective synthesis of mitochondrial proteins by Chinese hamster ovary cells severely starved for various amino acids

Chinese hamster ovary (CHO) cells were subjected to severe amino acid starvation for histidine, leucine, methionine, asparagine, tyrosine, glutamine, valine, and lysine, using amino acid analogs or mutations in specific aminoacyl-tRNA synthetases. At protein synthetic rates of less than 5%, in all cases, the newly synthesized proteins were found on two- dimensional electrophoretic gels to consist of a few intensely labeled spots, with the exception of lysine. This pattern could also be produced by strong inhibition of cytoplasmic protein synthesis with cycloheximide, and was abolished by preincubation with the mitochondrial protein synthesis inhibitor chloramphenicol. It appears therefore that the spots represent mitochondrial protein synthesis and that animal cells must have separate aminoacyl-tRNA synthetases for mitochondrial tRNAs corresponding to all these amino acids except, possibly, for lysine.     

Mitochondrial tRNA gene mutations in patients having mitochondrial disease with lactic acidosis

Lactic acidosis has been associated with a variety of clinical conditions and can be due to mutation in nuclear or mitochondrial genes. We performed mutations screening of all mitochondrial tRNA genes in 44 patients who referred as hyperlactic acidosis. Patients showed heterogeneous phenotypes including Leigh disease in four, MELAS in six, unclassified mitochondrial myopathy in 10, cardiomyopathy in five, MERRF in one, pure lactic acidosis in six, and others in 12 including facio-scaplo-femoral muscular dystrophy (FSFD), familial cerebellar ataxia, recurrent Reye syndrome, cerebral palsy with mental retardation. We measured enzymatic activities of pyruvate dehydrogenase complex, and respiratory chain enzymes. All mitochondrial tRNA genes and known mutation of ATPase 6 were studied by single strand conformation polymorphism (SSCP), automated DNA sequence and PCR-RFLP methods. We have found one patient with PDHC deficiency and six patients with Complex I+IV deficiency, though the most of the patients showed subnormal to deficient state of respiratory chain enzyme activities. We have identified one of the nucleotide changes in 29 patients. Single nucleotide changes in mitochondrial tRNA genes are found in 27 patients and one in ATPase 6 gene in two patients. One of four pathogenic point mutations (A3243G, C3303T, A8348G, and T8993G) was identified in 12 patients who showed the phenotype of Leigh syndrome, MELAS, cardimyopathy and cerebral palsy with epilepsy. Seventeen patients have one of the normal polymorphisms in the mitochondrial tRNA gene reported before. SSCP and PCR-RFLP could detect the heteroplasmic condition when the percentage of mutant up to 5, however, it cannot be observed by direct sequencing method. It is important to screen the mtDNA mutation not only by direct sequence but also by PCR-RFLP and the other sensitive methods to detect the heroplasmy when lactic acidosis has been documented in the patients who are not fulfilled the criteria of mitochondrial disorders.     

Hypersensitivity of A8344G MERRF mutated cybrid cells to staurosporine-induced cell death is mediated by calcium-dependent activation of calpains

Mutations in the mitochondrial DNA can lead to the development of mitochondrial diseases such as Myoclonic Epilepsy with Ragged Red Fibers (MERRF) or Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS). We first show that human 143B-derived cybrid cells harboring either the A8344G (MERRF) or the A3243G (MELAS) mutation, are more prone to undergo apoptosis then their wild-type counterpart, when challenged with various apoptotic inducers such as staurosporine, etoposide and TRAIL. In addition, investigating the mechanisms underlying A8344G cybrid cells hypersensitivity to staurosporine-induced cell death, we found that staurosporine treatment activates caspases independently of cytochrome c release in both wild-type and mutated cells. Caspases are activated, at least partly, through the activation of calcium-dependent calpain proteases, a pathway that is more strongly activated in mutated cybrid cells than in wild-type cells exposed to staurosporine. These results suggest that calcium homeostasis perturbation induced by mitochondrial dysfunction could predispose cells to apoptosis, a process that could take part into the progressive cell degeneration observed in MERRF syndrome, and more generally in mitochondrial diseases.     

Age related profiles of free amino acids in human skeletal muscle

Sarcopenia describes the involuntary decline in muscle mass with aging, coupled with fatigue, and loss of force and function. We investigated 113 human muscle biopsy specimens obtained from patients with neuromuscular diseases and controls. We measured 21 amino acids in these muscle biopsies. Age emerged as a significant negative predictor of cytosolic concentration ratio of glutamine to total branched chain amino acids and of glutamine to total aromatic amino acids using stepwise multiple linear regression analysis. This pattern of alteration corresponds well to documented alterations in skeletal muscle of critically ill patients and after immobilization. Additionally, in myositis, citrulline was significantly elevated, while glutamate, lysine and taurine were significantly reduced. Furthermore, in sporadic amyotrophic lateral sclerosis (sALS) the total aromatic amino acids, arginine, glutamate, threonine, and tyrosine were significantly elevated. This study provides evidence, that alteration of glutamine is correlated to aging and might reflect increased proteolysis in aged and diseased human skeletal muscle.     

Neuropathology of mitochondrial diseases

The term “mitochondrial diseases” (MD) refers to a group of disorders related to respiratory chain dysfunction. Clinical features are usually extremely heterogeneous because MD may involve several tissues with different degrees of severity. Muscle and brain are mostly affected, probably because of their high dependence on oxidative metabolism. Muscle can be the only affected tissue or involved as a part of a multi-system disease; ragged red fibers, accumulation of structurally altered mitochondria and cytochrome-c-oxidase (COX) negative fibers are the main pathological features. In mitochondrial encephalopathies, central nervous system (CNS) structures are affected according to different patterns of distribution and severity. Characteristic lesions are neuronal loss, vasculo-necrotic changes, gliosis, demyelination and spongy degeneration. In accordance with either grey matter or white matter involvement two main groups of diseases may be distinguished. Neuronal loss and vasculo-necrotic multifocal lesions are the common features of grey matter involvement; demyelination and spongy degeneration occur when white matter is affected, often associated with less severe lesions of the grey structures. Grey matter lesions are prevalent in MERRF, MELAS, Alpers and Leigh syndromes. White matter involvement is always seen in Kearns-Sayre syndrome and was recently described in mtDNA depletion syndrome linked to dGK mutations and in the rare conditions associated with complex I and II deficiency. In this review we describe the main histopathological features of muscle and CNS lesions in mitochondrial diseases.     

Genetic system for maintaining the mitochondrial human genome in yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.     

Taurine-containing uridine modifications in tRNA anticodons are required to decipher non-universal genetic codes in ascidian mitochondria

Variations in the genetic code are found frequently in mitochondrial decoding systems. Four non-universal genetic codes are employed in ascidian mitochondria: AUA for Met, UGA for Trp, and AGA/AGG(AGR) for Gly. To clarify the decoding mechanism for the non-universal genetic codes, we isolated and analyzed mitochondrial tRNAs for Trp, Met, and Gly from an ascidian, Halocynthia roretzi. Mass spectrometric analysis identified 5-taurinomethyluridine (τm(5)U) at the anticodon wobble positions of tRNA(Met)(AUR), tRNA(Trp)(UGR), and tRNA(Gly)(AGR), suggesting that τm(5)U plays a critical role in the accurate deciphering of all four non-universal codes by preventing the misreading of pyrimidine-ending near-cognate codons (NNY) in their respective family boxes. Acquisition of the wobble modification appears to be a prerequisite for the genetic code alteration.     

Avoidance of antisense, antiterminator tRNA anticodons in vertebrate mitochondria

Protein synthesis (translation) stops at stop codons, codons not complemented by tRNA anticodons. tRNAs matching stops, antitermination (Ter) tRNAs, prevent translational termination, producing dysfunctional proteins. Genomes avoid tRNAs with anticodons whose complement (the anticodon of the ‘antisense’ tRNA) matches stops. This suggests that antisense tRNAs, which also form cloverleaves, are occasionally expressed. Mitochondrial antisense tRNA expression is plausible, because both DNA strands are transcribed as single RNAs, and tRNA structures signal RNA maturation. Results describe potential antisense Ter tRNAs in mammalian mitochondrial genomes detected by tRNAscan-SE, and evidence for adaptations preventing translational antitermination: genomes possessing Ter tRNAs use less corresponding stop codons; antisense Ter tRNAs form weaker cloverleaves than homologuous non-Ter antisense tRNAs; and genomic stop codon usages decrease with stabilities of codon-anticodon interactions and of Ter tRNA cloverleaves. This suggests that antisense tRNAs frequently function in translation. Results suggest that opposite strand coding is exceptional in modern genes, yet might be frequent for mitochondrial tRNAs. This adds antisense tRNA templating to other mitochondrial tRNA functions: sense tRNA templating, formation and regulation of secondary (light strand DNA) replication origins. Antitermination probably affects mitochondrial degenerative diseases and ageing: pathogenic mutations are twice as frequent in tRNAs with antisense Ter anticodons than in other tRNAs, and species lacking mitochondrial antisense Ter tRNAs have longer mean maximal lifespans than those possessing antisense Ter tRNAs.     

Extracellular Levels of Amino Acids and Choline in Human High Grade Gliomas: An Intraoperative Microdialysis Study

The concentrations of endogenous amino acids and choline in the extracellular fluid of human cerebral gliomas have been measured, for the first time, by in vivo microdialysis. Glioblastoma growth was associated with increased concentrations of choline, GABA, isoleucine, leucine, lysine, phenylalanine, taurine, tyrosine, and valine. There was no difference between grade III and grade IV tumors in the concentrations of phenylalanine, isoleucine, tyrosine, valine, and lysine, whereas the concentrations of choline, aspartate, taurine, GABA, leucine, and glutamate were significantly different in the two tumor-grade subgroups. In contrast to the other compounds, the concentration of glutamate was decreased in glioma. The parenchyma adjacent to the tumor showed significant changes only in the extracellular concentration of glutamate, isoleucine, and valine. The concentrations of choline and the amino acids, glutamate, leucine, taurine, and tyrosine showed significant positive correlations with the degree of cell proliferation. Epilepsy, which is relatively common in subjects with gliomas, was shown to be a significant confounding variable when the extracellular concentrations of aspartate, glutamate and GABA were considered.