Biochimica et biophysica acta最新文献

The availability of monoclonal antibodies (mAbs) against the proteins of the oxidative phosphorylation chain (OXPHOS) and other mitochondrial components facilitates the analysis and ultimately the diagnosis of mitochondrially related diseases. mAbs against each of the five complexes and pyruvate dehydrogenase (PDH) are the basis of a rapid and simple immunocytochemical approach [Hanson, B.J., Capaldi, R.A., Marusich, M.F. and Sherwood, S.W., J. Histochem. Cytochem. 50 (2002) 1281-1288]. This approach can be used to detect if complexes have altered assembly in mitochondrial disease due to mutations in nuclear encoded genes, such as in Leigh's disease, or in mitochondrially encoded genes, e.g., MELAS. Other mAbs have recently been obtained that can immunocapture each of the five OXPHOS complexes, PDH and the adenine nucleotide translocase (ANT) from very small amounts of tissue such as that obtained from cell culture or needle biopsies from patients. When adapted to a 96-well plate format, these mAbs allow measurement of the specific activity of each of the mitochondrial components individually and analysis of their subunit composition and state of posttranslational modification. The immunocapture protocol should be useful not only in the analysis of genetic mitochondrial diseases but also in evaluating and ultimately diagnosing late-onset mitochondrial disorders including Parkinson's disease, Alzheimer's disease, and late-onset diabetes, which are thought to result from accumulated oxidative damage to mitochondrial proteins such as the OXPHOS chain.

After reviewing the history of mitochondrial diseases, I follow a genetic classification to discuss new developments and old conundrums. In the field of mitochondrial DNA (mtDNA) mutations, I argue that we are not yet scraping the bottom of the barrel because: (i) new mtDNA mutations are still being discovered, especially in protein-coding genes; (ii) the pathogenicity of homoplasmic mutations is being revisited; (iii) some genetic dogmas are chipped but not broken; (iv) mtDNA haplotypes are gaining interest in human pathology; (v) pathogenesis is still largely enigmatic. In the field of nuclear DNA (nDNA) mutations, there has been good progress in our understanding of disorders due to faulty intergenomic communication. Of the genes responsible for multiple deletions and depletion of mtDNA, mutations in POLG have been associated with a great variety of clinical phenotypes in humans and to precocious aging in mice. Novel pathogenetic mechanisms include alterations in the lipid milieu of the inner mitochondrial membrane and mutations in genes controlling mitochondrial motility, fission, and fusion.

The work from our laboratory on complex I-deficient Chinese hamster cell mutants is reviewed. Several complementation groups with a complete defect have been identified. Three of these are due to X-linked mutations, and the mutated genes for two have been identified. We describe null mutants in the genes for the subunits MWFE (gene: NDUFA1) and ESSS. They represent small integral membrane proteins localized in the Ialpha (Igamma) and Ibeta subcomplexes, respectively [J. Hirst, J. Carroll, I.M. Fearnley, R.J. Shannon, J.E. Walker. The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim. Biophys. Acta 1604 (7-10-2003) 135-150.]. Both are absolutely essential for assembly and activity of complex I. Epitope-tagged versions of these proteins can be expressed from a poly-cistronic vector to complement the mutants, or to be co-expressed with the endogenous proteins in other hamster cell lines (mutant or wild type), or human cells. Structure-function analyses can be performed with proteins altered by site-directed mutagenesis. A cell line has been constructed in which the MWFE subunit is conditionally expressed, opening a window on the kinetics of assembly of complex I. Its targeting, import into mitochondria, and orientation in the inner membrane have also been investigated. The two proteins have recently been shown to be the targets for a cAMP-dependent kinase [R. Chen, I.M. Fearnley, S.Y. Peak_Chew, J.E. Walker. The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. xx (2004) xx-xx.]. The epitope-tagged proteins can be cross-linked with other complex I subunits.

Whether regular exercise is beneficial or should be avoided is a question currently unsettled in patients with heteroplasmic mitochondrial DNA (mtDNA) disorders of skeletal muscle. Deleterious effects of habitual physical inactivity superimposed upon impaired mitochondrial oxidative phosphorylation may contribute to varying degrees of exercise intolerance in these patients. Endurance exercise training is widely known to improve exercise capacity in healthy subjects and various chronic-disease patient populations. Although we have shown that beneficial physiological and biochemical responses to training increase exercise tolerance in patients with mtDNA defects, knowledge of the muscle adaptive response to endurance training within the setting of mitochondrial heteroplasmy remains limited. In order to determine advisability of endurance training as therapy, it remains to be established whether potential endurance training-induced increases in mutant mtDNA levels may be offset by increases in absolute wild-type mtDNA levels, and whether chronic inactivity leads to a selective down-regulation of wild-type mtDNA. Resistance training utilizes a different adaptive exercise approach to induce the transfer of normal mitochondrial templates from satellite cells to mature muscle fibers of patients with sporadic mtDNA disorders. The efficacy and safety of this approach needs to be further established. Our current inability to clearly advise patients to "use it or lose it" underscores the immediate urgency of studying the effects of exercise on skeletal muscle of patients with heteroplasmic mtDNA defects.

Initiation of transcription at mitochondrial promoters in mammalian cells requires the simultaneous presence of a monomeric mitochondrial RNA polymerase, mitochondrial transcription factor A, and either transcription factor B1 or B2. We here review recent progress in our understanding of how these basal factors cooperate in the initiation and regulation of mitochondrial transcription. We describe the evolutionary origin of individual transcription factors and discuss how these phylogenetic relationships may facilitate a molecular understanding of the mitochondrial transcription machinery.

Biochemical diagnosis of mitochondrial respiratory chain disorders requires caution to avoid misdiagnosis of secondary enzyme defects, and can be improved by the use of conservative diagnostic criteria. Pathogenic mutations causing mitochondrial disorders have now been identified in more than 30 mitochondrial DNA (mtDNA) genes encoding respiratory chain subunits, ribosomal- and t-RNAs. mtDNA mutations appear to be responsible for most adult patients with mitochondrial disease and approximately a quarter of paediatric patients. A family history suggesting maternal inheritance is the exception rather than the norm for children with mtDNA mutations, many of whom have de novo mutations. Prenatal diagnosis and pre-implantation genetic diagnosis can be offered to some women at risk of transmitting a mtDNA mutation, particularly those at lower recurrence risk. Mutations in more than 30 nuclear genes, including those encoding for respiratory chain subunits and assembly factors, have now been shown to cause mitochondrial disorders, creating difficulties in prioritising which genes should be studied by mutation analysis in individual patients. A number of approaches offer promise to guide the choice of candidate genes, including Blue Native-PAGE immunoblotting and microarray expression analysis.

The mitochondrial respiratory chain (RC) results from the expression of both mitochondrial and nuclear genes. The number of disease-causing mutations in nuclear genes is steadily growing and mitochondrial DNA (mtDNA) deletions and mutations account for no more than 15-20% of pediatric patients. Unfortunately, the disease-causing mutations have been identified for only a small number of patients. Thus, elucidating the genetic bases of RC is both essential for genetic diagnosis of patients and for fundamental knowledge of these disorders. The molecular diagnostics of mitochondrial disorders come under both genetic diagnosis and research. Indeed, identification of a new gene in a specific patient allows to perform genetic diagnosis in other families and identification of mutations in already known disease-causing genes allows to constitute a cohort of patients for further functional studies. Thus, elucidating the genetic bases of RC deficiency is an essential task that needs the use of several appropriate strategies. Fine phenotypage of patients and candidate gene screening is the first step for the constitution of a well-characterized cohort of patients. Genetic mapping has to be used in large families. This approach is greatly enhanced in the case of consanguineous families. The consanguinity of the parents should also lead to test genetic markers surrounding the gene loci rather than to directly sequence several candidate genes. However, the main problem is encountered in the cases of sporadic cases for which no genetic approaches can be developed. In these cases, functional complementation by human chromosomes or cDNA is the only presently available strategy.

Mitochondria are eukaryotic organelles that originated from a single bacterial endosymbiosis some 2 billion years ago. The transition from the ancestral endosymbiont to the modern mitochondrion has been accompanied by major changes in its protein content, the so-called proteome. These changes included complete loss of some bacterial pathways, amelioration of others and gain of completely new complexes of eukaryotic origin such as the ATP/ADP translocase and most of the mitochondrial protein import machinery. This renewal of proteins has been so extensive that only 14-16% of modern mitochondrial proteome has an origin that can be traced back to the bacterial endosymbiont. The rest consists of proteins of diverse origin that were eventually recruited to function in the organelle. This shaping of the proteome content reflects the transformation of mitochondria into a highly specialized organelle that, besides ATP production, comprises a variety of functions within the eukaryotic metabolism. Here we review recent advances in the fields of comparative genomics and proteomics that are throwing light on the origin and evolution of the mitochondrial proteome.

Defects of the mitochondrial genome are a significant cause of disease. Patients suffer from a wide variety of clinical presentations, ranging from fatal infantile disease to mild muscle weakness. Most disorders, however, are characterized by inexorable progression. As mutations often cause defects in several components of the complexes that couple oxidative phosphorylation, this terminal state of oxidative metabolism cannot be readily bypassed by dietary means, leading to the search for novel therapies. In this article, we present the theory behind several concepts and report progress. We also discuss some of the recent difficulties encountered in the progress towards an antigenomc approach to treating mtDNA disorders.