Screening human EST database for identification of candidate genes in respiratory chain deficiency

Disorders of mitochondrial oxidative phosphorylation (OXPHOS) are now recognized as major causes of human metabolic diseases and several mutations of mitochondrial and nuclear genes encoding respiratory chain components have been reported. Interestingly, mutations of nuclear genes involved in mitochondrial respiratory chain assembly, protein trafficking, and iron metabolism are also known to alter oxidative phosphorylation. While several hundred of these genes have been described in yeast, only a few nuclear genes have been hitherto identified in humans. Yeast gene databases present therefore an invaluable tool for identification of human homologues that should be regarded as candidate genes in OXPHOS diseases. In an attempt to identify the human counterparts of yeast genes, we developed a systematic comparison of yeast protein sequences to the GenBank dbEST database. Starting from 340 yeast protein sequences as templates, we searched the human dbEST counterparts using the BLAST similarity searching program and identified 102 groups of human EST likely to represent orthologues of yeast genes because of significant homology. This collection of human genes possibly related to mitochondrial OXPHOS may help identify nuclear genes responsible of mitochondrial disorders.     

Mitochondria and degenerative disorders

In mammalian cells, mitochondria provide energy from aerobic metabolism. They play an important regulatory role in apoptosis, produce and detoxify free radicals, and serve as a cellular calcium buffer. Neurodegenerative disorders involving mitochondria can be divided into those caused by oxidative phosphorylation (OXPHOS) abnormalities either due to mitochondrial DNA (mtDNA) abnormalities, e.g., chronic external ophthalmoplegia, or due to nuclear mutations of OXPHOS proteins, e.g., complex I and II associated with Leigh syndrome. There are diseases caused by nuclear genes encoding non-OXPHOS mitochondrial proteins, such as frataxin in Friedreich ataxia (which is likely to play an important role in mitochondrial-cytosolic iron cycling), paraplegin (possibly a mitochondrial ATP-dependent zinc metalloprotease of the AAA-ATPases in hereditary spastic paraparesis), and possibly Wilson disease protein (an abnormal copper transporting ATP-dependent P-type ATPase associated with Wilson disease). Huntingon disease is an example of diseases with OXPHOS defects associated with mutations of nuclear genes encoding non-mitochondrial proteins such as huntingtin. There are also disorders with evidence of mitochondrial involvement that cannot as yet be assigned. These include Parkinson disease (where a complex I defect is described and free radicals are generated from dopamine metabolism), amyotrophic lateral sclerosis, and Alzheimer disease, where there is evidence to suggest mitochondrial involvement perhaps secondary to other abnormalities.     

Occurrence of nuclear-encoded tRNAIle in mitochondria of the liverwort Marchantia polymorpha

 Although 29 tRNA genes have been deduced from the complete nucleotide sequence of the mitochondrial genome from the liverwort Marchantia polymorpha, a tRNA^Ile gene decoding AUU and AUC codons is conspicuously absent. In order to address the question of the possible involvement of nuclear-encoded tRNA, we isolated and identified three variant copies of the nuclear-encoded tRNA^Ile(AAU) gene from the liverwort. Northern analysis showed the presence of nuclear-encoded tRNA^Ile both in the mitochondrion and the cytosol, while both chloroplast DNA-encoded tRNA^Ile and nuclear-encoded tRNA^Tyr were absent in liverwort mitochondria. These results unequivocally establish that import of nuclear tRNA^Ile into mitochondria indeed occurs in one of the most primitive plants, M. polymorpha. Received: 11 January 1996/18 March 1996     

Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency

NADH-ubiquinone oxidoreductase (complex I) deficiency is amongst the most encountered defects of the mitochondrial oxidative phosphorylation (OXPHOS) system and is associated with a wide variety of clinical signs and symptoms. Mutations in complex I nuclear structural genes are the most common cause of isolated complex I enzyme deficiencies. The cell biological consequences of such mutations are poorly understood. In this paper we have used blue native electrophoresis in order to study how different nuclear mutations affect the integrity of mitochondrial OXPHOS complexes in fibroblasts from 15 complex I-deficient patients. Our results show an important decrease in the levels of intact complex I in patients harboring mutations in nuclear-encoded complex I subunits, indicating that complex I assembly and/or stability is compromised. Different patterns of low molecular weight subcomplexes are present in these patients, suggesting that the formation of the peripheral arm is affected at an early assembly stage. Mutations in complex I genes can also affect the stability of other mitochondrial complexes, with a specific decrease of fully-assembled complex III in patients with mutations in NDUFS2 and NDUFS4. We have extended this analysis to patients with an isolated complex I deficiency in which no mutations in structural subunits have been found. In this group, we can discriminate between complex I assembly and catalytic defects attending to the fact whether there is a correlation between assembly/activity levels or not. This will help us to point more selectively to candidate genes for pathogenic mutations that could lead to an isolated complex I defect.     

Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells

Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly.     

Nuclear DNA origin of cytochrome c oxidase deficiency in Leigh's syndrome: genetic evidence based on patient's-derived rho{degrees} transformants

Defects of the respiratory chain carrying out oxidative phosphorylation(OXPHOS) are the biochemical hallmark of human mitochondrialdisorders. Faulty OXPHOS can be due to mutations in either nuclearor mitochondrial genes, that are involved in the synthesis ofindividual respiratory subunits or in their post-translationalcontrol. The most common mitochondrial disorder of infancy andchildhood is Leigh's syndrome, a severe encephalopathy, oftenassociated with a defect of cytochrome c oxidase (COX). In orderto demonstrate which genome is primarily involved in COX-deficient(COX(^–))-Leigh's syndrome, we generated two lines of transmitochondrialcybrids. The first was obtained by fusing nuclear DNA-less cytoplastsderived from normal fibroblasts, with mitochondrial DNA-less(rho°) transformant fibroblasts derived from a patient withCOX(^–)-Leigh's syndrome. The second cybrid line was obtainedby fusing rho° cells derived from 143B.TK^– human osteosarcomacells, with cytoplasts derived from the same patient. The firstcybrid line showed a specific and severe COX(-) phenotype, whilein the second all the respiratory chain complexes, includingCOX, were normal. These results indicate that the COX defectin our patient is due to a mutation of a nuclear gene. The useof cybrids obtained from ‘customized’, patient-derivedrho° cells can have wide applications in the identificationof respiratory chain defects originated by nuclear DNA—encodedmutations, and in the study of nuclear DNA-mitochondrial DNAinteractions.     

ECHS1 Mutations Cause Combined Respiratory Chain Deficiency Resulting in Leigh Syndrome

The human ECHS1 gene encodes the short‐chain enoyl coenzyme A hydratase, the enzyme that catalyzes the second step of β‐oxidation of fatty acids in the mitochondrial matrix. We report on a boy with ECHS1 deficiency who was diagnosed with Leigh syndrome at 21 months of age. The patient presented with hypotonia, metabolic acidosis, and developmental delay. A combined respiratory chain deficiency was also observed. Targeted exome sequencing of 776 mitochondria‐associated genes encoded by nuclear DNA identified compound heterozygous mutations in ECHS1. ECHS1 protein expression was severely depleted in the patient's skeletal muscle and patient‐derived myoblasts; a marked decrease in enzyme activity was also evident in patient‐derived myoblasts. Immortalized patient‐derived myoblasts that expressed exogenous wild‐type ECHS1 exhibited the recovery of the ECHS1 activity, indicating that the gene defect was pathogenic. Mitochondrial respiratory complex activity was also mostly restored in these cells, suggesting that there was an unidentified link between deficiency of ECHS1 and respiratory chain. Here, we describe the patient with ECHS1 deficiency; these findings will advance our understanding not only the pathology of mitochondrial fatty acid β‐oxidation disorders, but also the regulation of mitochondrial metabolism.