Mitochondrial diseases

By convention, the term "mitochondrial diseases" refers to disorders of the mitochondrial respiratory chain, which is the only metabolic pathway in the cell that is under the dual control of the mitochondrial genome (mtDNA) and the nuclear genome (nDNA). Therefore, a genetic classification of the mitochondrial diseases distinguishes disorders due to mutations in mtDNA, which are governed by the relatively lax rules of mitochondrial genetics, and disorders due to mutations in nDNA, which are governed by the stricter rules of mendelian genetics. Mutations in mtDNA can be divided into those that impair mitochondrial protein synthesis in toto and those that affect any one of the 13 respiratory chain subunits encoded by mtDNA. Essential clinical features for each group of diseases are reviewed. Disorders due to mutations in nDNA are more abundant not only because most respiratory chain subunits are nucleus-encoded but also because correct assembly and functioning of the respiratory chain require numerous steps, all of which are under the control of nDNA. These steps (and related diseases) include: (i) synthesis of assembly proteins; (ii) intergenomic signaling; (iii) mitochondrial importation of nDNA-encoded proteins; (iv) synthesis of inner mitochondrial membrane phospholipids; (v) mitochondrial motility and fission.     

Mrpl36 Is Important for Generation of Assembly Competent Proteins during Mitochondrial Translation

The complexes of the respiratory chain represent mosaics of nuclear and mitochondrially encoded components. The processes by which synthesis and assembly of the various subunits are coordinated remain largely elusive. During evolution, many proteins of the mitochondrial ribosome acquired additional domains pointing at specific properties or functions of the translation machinery in mitochondria. Here, we analyzed the function of Mrpl36, a protein associated with the large subunit of the mitochondrial ribosome. This protein, homologous to the ribosomal protein L31 from bacteria, contains a mitochondria-specific C-terminal domain that is not required for protein synthesis per se; however, its absence decreases stability of Mrpl36. Cells lacking this C-terminal domain can still synthesize proteins, but these translation products fail to be properly assembled into respiratory chain complexes and are rapidly degraded. Surprisingly, overexpression of Mrpl36 seems to even increase the efficiency of mitochondrial translation. Our data suggest that Mrpl36 plays a critical role during translation that determines the rate of respiratory chain assembly. This important function seems to be carried out by a stabilizing activity of Mrpl36 on the interaction between large and small ribosomal subunits, which could influence accuracy of protein synthesis.     

Nuclear genes and mitochondrial translation: a new class of genetic disease

Mitochondria contain a separate protein-synthesis machinery to produce the polypeptides encoded in mitochondrial DNA (mtDNA), and many mtDNA disease mutations affect this machinery. In humans, the mitochondrial rRNAs and tRNAs are encoded by mtDNA, whereas all proteins involved in mitochondrial translation are encoded by nuclear genes. Recently, several articles have discussed the identification of pathological mutations in nuclear genes encoding components of this protein-synthesis machinery, suggesting that these types of mutation are a frequent cause of human genetic diseases.     

Coupling of mitochondrial translation with the formation of respiratory complexes in yeast mitochondria

In contrast to most other eukaryotic organisms, yeast can survive without respiration. This ability has been exploited to investigate nuclear genes required for expression of mitochondrial DNA. Availability of complete Saccharomyces cerevisiae genomic sequence has provided additional help in detailed molecular analysis. Seven of the eight major products encoded by mitochondrial DNA are hydrophobic subunits of respiratory complexes in the inner membrane. Localization of the translation process in the same cellular compartment ensures synthesis of mitochondrially encoded proteins near sites of their assembly into multimeric respiratory complexes. Association of mitochondrial ribosomes with the membrane is mediated by mRNA-specific translational activators, that are involved in the recognition of initiation codon. The newly synthesized mitochondrial proteins are transferred to membrane by a specific export system. This review discusses the role of membrane-localized factors responsible for quality control and turnover of mitochondrially synthesized subunits as well as for assembly of respiratory complexes.     

A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies

Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.     

Regulation of biosynthesis of the rat liver inner mitochondrial membrane by thyroid hormone

Regulation of mitochondrial protein synthesis by thyroid hormone has been studied in isolated rat hepatocytes and liver mitochondria. Small doses (5 micrograms/100 g body wt) of triiodothyronine (T3) injected into hypothyroid rats increased both state 3 and 4 respiration by approximately 100%, while the ADP:O ratio remained constant. This suggests that T3 increases the numbers of functional respiratory chain units. T3 also induces mitochondrial protein synthesis by 50-100%. Analysis of the mitochondrial translation products show that all of the products were induced. No differential translation of the peptides involved in the respiratory chain was found. Regulation of the cytoplasmically made inner membrane peptides was also investigated in isolated hepatocytes. The majority of these peptides were not influenced by T3, in contrast to the finding with mitochondrial translation products. Those found to be regulated by T3 belong to two subsets, which were either induced or repressed by hormone. Thus, T3 stimulated a general increase in the synthesis of mitochondrially translated inner membrane peptides, but regulates selectively those inner membrane peptides translated on cytoplasmic ribosomes. The findings suggest that hormone regulation of the respiratory chain is exerted through a few selective proteins, perhaps those which require subunits made from both nuclear and mitochondrial genes.     

Clingy genes: Why were genes for ribosomal proteins retained in many mitochondrial genomes?

Why mitochondria still retain their own genome is a puzzle given the enormous effort to maintain a mitochondrial translation machinery. Most mitochondrially encoded proteins are membrane-embedded subunits of the respiratory chain. Their hydrophobicity presumably impedes their import into mitochondria. However, many mitochondrial genomes also encode protein subunits of the mitochondrial ribosome. These proteins lack transmembrane domains and hydrophobicity cannot explain why their genes remained in mitochondria. In this review, we provide an overview about mitochondrially encoded subunits of mitochondrial ribosomes of fungi, plants and protists. Moreover, we discuss and evaluate different hypotheses which were put forward to explain why (ribosomal) proteins remained mitochondrially encoded. It seems likely that the synthesis of ribosomal proteins in the mitochondrial matrix is used to regulate the assembly of the mitochondrial ribosome within mitochondria and to avoid problems that mitochondrial proteins might pose for cytosolic proteostasis and for the assembly of cytosolic ribosomes.     

The yeast nuclear gene MRF1 encodes a mitochondrial peptide chain release factor and cures several mitochondrial RNA splicing defects.

We report the molecular cloning, sequencing and genetic characterization of the first gene encoding an organellar polypeptide chain release factor, the MRF1 gene of the yeast Saccharomyces cerevisiae. The MRF1 gene was cloned by genetic complementation of a respiratory deficient mutant disturbed in the expression of the mitochondrial genes encoding cytochrome c oxidase subunit 1 and 2, COX1 and COX2. For COX1 this defect has been attributed to an impaired processing of several introns. Sequence analysis of the MRF1 gene revealed that it encodes a protein highly similar to prokaryotic peptide chain release factors, especially RF-1. Disruption of the gene results in a high instability of the mitochondrial genome, a hallmark for a strict lesion in mitochondrial protein synthesis. The respiratory negative phenotype of mrf1 mutants lacking all known mitochondrial introns and the reduced synthesis of mitochondrial translation products encoded by unsplit genes confirm a primary defect in mitochondrial protein synthesis. Over-expression of the MRF1 gene in a mitochondrial nonsense suppressor strain reduces suppression in a dosage-dependent manner, shedding new light on the role of the '530 region' of 16S-like ribosomal RNA in translational fidelity.     

线粒体核糖体蛋白质与人类恶性肿瘤

线粒体拥有自身独特的核糖体--线粒体核糖体,用于翻译线粒体DNA（mitochondrial DNA, mtDNA）编码的基因。线粒体核糖体由核基因编码的线粒体核糖体蛋白质(mitochondrial ribosomal protein, MRPs)和线粒体自身编码的rRNA组装而成。MRPs表达失调会引发代谢紊乱、呼吸链受损,导致细胞发生功能障碍和异常增殖,甚至发生癌变等恶性转化。大量研究证明,MRPs在不同的肿瘤细胞中表达异常,提示着MRPs在肿瘤发生发展过程中发挥着重要作用。本文就线粒体核糖体蛋白质与人类恶性肿瘤发生的关系作一综述,为进一步阐明其在恶性肿瘤发生过程中的作用机制奠定基础。     

Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders.

More than 70 different point mutations in human mitochondrial tRNA genes are correlated with severe disorders, including fatal cardiopathies, encephalopathies, myopathies, and others. So far, investigation of the molecular impact(s) of mutations has focused on the affected tRNA itself by seeking structural and/or functional perturbations capable of interfering with synthesis of the 13 mitochondrion-encoded subunits of respiratory chain complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Analysis of several hundred mitochondrial proteins from sibling cybrid cell lines by two-dimensional electrophoresis, an approach that takes into account all regulatory steps of mitochondrial and nuclear gene expression, indeed reveals a number of up- and downregulated proteins when healthy and single-point-mutation-carrying mitochondria representative of either MELAS or MERRF syndrome were compared. Assignment by mass spectrometry of the two proteins which exhibit obvious large quantitative decreases in the levels of both pathologic mitochondria identified nuclear-encoded subunits of cytochrome c oxidase, a respiratory chain complex. This clearly shows a linkage between the effects of mutations in mitochondrial tRNA genes and the steady-state level of nuclear-encoded proteins in mitochondria. It opens new routes toward a large-scale exploration of potential proteic partners involved in the genotype-phenotype correlation of mitochondrial disorders.     

The diabetes-associated 3243 mutation in the mitochondrial tRNA(Leu(UUR)) gene causes severe mitochondrial dysfunction without a strong decrease in protein synthesis rate.

Cells harboring patient-derived mitochondria with an A-to-G transition at nucleotide position 3243 of their mitochondrial DNA display severe loss of respiration when compared with cells containing the wild-type adenine but otherwise identical mitochondrial DNA sequence. The amount and degree of leucylation of tRNA(Leu(UUR)) were both found to be highly reduced in mutant cells. Despite the low level of leucyl-tRNA(Leu(UUR)), the rate of mitochondrial translation was not seriously affected by this mutation. Therefore, decrease of mitochondrial protein synthesis as such does not appear to be a necessary prerequisite for loss of respiration. Rather, the mitochondrially encoded proteins seem subject to elevated degradation, leading to a severe reduction in their steady state levels. Our results favor a scheme in which the 3243 mutation causes loss of respiration through accelerated protein degradation, leading to a disequilibrium between the levels of mitochondrial and nuclear encoded respiratory chain subunits and thereby a reduction of functional respiratory chain complexes. The possible mechanisms underlying the pathogenesis of mitochondrial diabetes is discussed.     

Mechanism of protein biosynthesis in mammalian mitochondria

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

The relationship of cytoplasmic and mitochondrial protein synthesis in yeasts: the mapping of mitochondrial mutations neutralizing the exhibition of nuclear omnipotent suppressors

The genetic and physical mapping of mitochondrial mutations [CRD] neutralizing respiratory deficiency in sup1 and sup2 mutants was performed. The genetical methods demonstrated improbability of location of these mutations in genes coding for the enzymes of respiratory chain and for 21S rRNA. Southern-blot analysis has shown these mutations to be localized in the Hinc10 fragment of mitochondrial genome. This fact was interpreted as indication that var1 gene is affected by [CRD] mutations. Our results are in agreement with the hypothesis on the participation of sup1(2) proteins in mitochondrial translation.     

Insertion of proteins into the inner membrane of mitochondria: the role of the Oxa1 complex

The inner mitochondrial membrane harbors a large number of proteins that display a wide range of topological arrangements. The majority of these proteins are encoded in the cell's nucleus, but a few polytopic proteins, all subunits of respiratory chain complexes are encoded by the mitochondrial genome. A number of distinct sorting mechanisms exist to direct these proteins into the mitochondrial inner membrane. One of these pathways involves the export of proteins from the matrix into the inner membrane and is used by both proteins synthesized within the mitochondria, as well as by a subset of nuclear encoded proteins. Prior to embarking on the export pathway, nuclear encoded proteins using this sorting route are initially imported into the mitochondrial matrix from the cytosol, their site of synthesis. Protein export from the matrix into the inner membrane bears similarities to Sec-independent protein export in bacteria and requires the function of the Oxa1 protein. Oxa1 is a component of a general protein insertion site in yeast mitochondrial inner membrane used by both nuclear and mitochondrial DNA encoded proteins. Oxa1 is a member of the conserved Oxa1/YidC/Alb3 protein family found throughout prokaryotes throughout eukaryotes (where it is found in mitochondria and chloroplasts). The evidence to demonstrate that the Oxa1/YidC/Alb3 protein family represents a novel evolutionarily conserved membrane insertion machinery is reviewed here.     

Proteomic consequences of a human mitochondrial tRNA mutation beyond the frame of mitochondrial translation

Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.     

MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome

Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome.     

The site of synthesis of the iron-sulfur subunits of the flavoprotein and iron-protein fractions of human NADH dehydrogenase

The site of synthesis of the iron-sulfur subunits of the flavoprotein and iron-protein fractions of the human respiratory chain NADH dehydrogenase has been investigated to test the possibility that any of them is synthesized in mitochondria. For this purpose, antibodies specific for individual subunits of the bovine enzyme, which cross-reacted with the homologous human subunits in immunoblot assays, were tested against HeLa cell mitochondrial proteins labeled in vivo with [35S]methionine in the absence or presence of inhibitors of mitochondrial or cytoplasmic protein synthesis. The results clearly indicated that all the iron-sulfur subunits of the flavoprotein and iron-protein fractions of human complex I are synthesized in the cytosol and are, therefore, encoded in nuclear genes.