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Biomedical subjects

V Tiranti

Publications and source records attributed to V Tiranti.

At least 37 records · Page 2Linked to original sources
PubMed1999

Characterization of SURF-1 expression and Surf-1p function in normal and disease conditions.

Loss-of-function mutations of the SURF-1 gene have been associated with Leigh syndrome with cytochrome c oxidase (COX) deficiency. Mature Surf-1 protein (Surf-1p) is a 30 kDa hydrophobic polypeptide whose function is still unknown. Using antibodies against a recombinant, hemagglutinin-tagged Surf-1p, we have demonstrated that this protein is imported into mitochondria as a larger precursor, which is then processed into the mature product by cleaving off an N-terminal leader polypeptide of approximately 40 amino acids. By using western blot analysis with specific antibodies, we showed that Surf-1p is localized in and tightly bound to the mitochondrial inner membrane. The same analysis revealed that no protein is present in cell lines harboring loss-of-function mutations of SURF-1, regardless of their type and position. Northern blot analysis showed the virtual absence of specific SURF-1 transcripts in different mutant cell lines. This result suggests that several mutations of SURF-1 are associated with severe mRNA instability. To understand better whether and which domains of the protein are essential for function, we generated several constructs with truncated or partially deleted SURF-1 cDNAs. None of these constructs, expressed into Surf-1p null mutant cells, were able to rescue the COX phenotype, suggesting that different regions of the protein are all essential for function. Finally, experiments based on blue native two-dimensional gel electrophoresis indicated that assembly of COX in Surf-1p null mutants is blocked at an early step, most likely before the incorporation of subunit II in the nascent intermediates composed of subunit I alone or subunit I plus subunit IV. However, detection of residual amounts of fully assembled complex suggests a certain degree of redundancy of this system.

Animals↗
PubMed1998

Identification and characterization of human cDNAs specific to BCS1, PET112, SCO1, COX15, and COX11, five genes involved in the formation and function of the mitochondrial respiratory chain.

We have successfully applied a strategy based on the "cyberscreening" of the expressed sequence tags database using yeast protein sequences as "probes" to identify the human gene orthologs to BCS1, COX15, PET112, COX11, and SCO1, five yeast genes involved in the biogenesis of the mitochondrial respiratory chain complexes. In yeast, BCS1 is involved mainly in the assembly of complex III, while the other genes appear to control the structure/function of cytochrome-c oxidase. Significant amino acid identity and similarity were demonstrated by comparison of the human with the corresponding yeast polypeptides. Sequence alignment revealed numerous colinear identical regions and the conservation of functional domains. Mitochondrial targeting of the human gene products, suggested by computer analysis of the protein sequences, was confirmed by an in vitro import and protease-protection assay. These data strongly suggest that the human gene products share similar or identical functions with their yeast homologues. Genes controlling the structure/function of the respiratory chain complexes are attractive candidates for human mitochondrial disorders such as Leigh disease. However, both sequence analysis and functional complementation assays on an index patient do not support an etiological role for any of these genes.

Alkyl and Aryl Transferases↗
PubMed1998

A novel mutation in the mitochondrial tRNA(Val) gene associated with a complex neurological presentation.

We describe a patient who presented with progressive ataxia, seizures, mental deterioration, mild myopathy, and hearing loss. A novel heteroplasmic G-to-A transition was found, affecting the acceptor stem of the mitochondrial (mt) tRNA(Val) gene. Mutant mtDNA was 67% of total mtDNA in the muscle of the proband and was also present at low levels in the muscle of his healthy mother. It was absent in all of the numerous control DNA samples that were tested. Analysis of single muscle fibers revealed a significantly greater level of mutant mtDNA in cytochrome c oxidase-negative fibers. Mutations of mtDNA may be responsible of neurological syndromes that, like the case reported here, are clinically puzzling, and lack typical "mitochondrial" clues, such as elevated levels of blood lactate, overt defects of the respiratory complexes, and clinically documented maternal inheritance.

Base Sequence↗
PubMed1998

Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency.

Leigh disease associated with cytochrome c oxidase deficiency (LD[COX-]) is one of the most common disorders of the mitochondrial respiratory chain, in infancy and childhood. No mutations in any of the genes encoding the COX-protein subunits have been identified in LD(COX-) patients. Using complementation assays based on the fusion of LD(COX-) cell lines with several rodent/human rho0 hybrids, we demonstrated that the COX phenotype was rescued by the presence of a normal human chromosome 9. Linkage analysis restricted the disease locus to the subtelomeric region of chromosome 9q, within the 7-cM interval between markers D9S1847 and D9S1826. Candidate genes within this region include SURF-1, the yeast homologue (SHY-1) of which encodes a mitochondrial protein necessary for the maintenance of COX activity and respiration. Sequence analysis of SURF-1 revealed mutations in numerous DNA samples from LD(COX-) patients, indicating that this gene is responsible for the major complementation group in this important mitochondrial disorder.

Animals↗
PubMed1998

Mitochondrial disorders.

Mitochondrial respiration, the most efficient metabolic pathway devoted to energy production, is at the crosspoint of 2 quite different genetic systems, the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA). The latter encodes a few essential components of the mitochondrial respiratory chain and has unique molecular and genetic properties that account for some of the peculiar features of mitochondrial disorders. However, the perpetuation, propagation, and expression of mtDNA, the majority of the subunits of the respiratory complexes, as well as a number of genes involved in their assembly and turnover, are contained in the nuclear genome. Although mitochondrial disorders have been known for more than 30 years, a major breakthrough in their understanding has come much later, with the discovery of an impressive, ever-increasing number of mutations of mitochondrial DNA. Partial deletions or duplications of mtDNA, or maternally inherited point mutations, have been associated with well-defined clinical syndromes. However, phenotypes transmitted as mendelian traits have also been identified. These include clinical entities defined on the basis of specific biochemical defects, and also a few autosomal dominant or recessive syndromes associated with multiple deletions or tissue-specific depletion of mtDNA. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogenous. They range from lesions of single tissues or structures, such as the optic nerve in Leber hereditary optic neuropathy or the cochlea in maternally inherited nonsyndromic deafness, to more widespread lesions including myopathies, encephalomyopathies, cardiopathies, or complex multisystem syndromes. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights in this rapidly expanding area of human pathology.

DNA, Mitochondrial↗
PubMed1997

Characterization and expression of the mouse endonuclease G gene.

Endonuclease G (Endo G) is a nuclease of prokaryotic lineage found in the mitochondria of vertebrates that has been suggested to play a role in mitochondrial DNA (mtDNA) replication. We have isolated and sequenced the entire mouse endo G gene, determined the limits of the mRNA, and mapped the promoter region. The coding sequence of the single copy gene is interrupted by two introns and analysis of the transcripts does not support a model by which more than one Endo G isoform could be produced by alternative splicing. We have also characterized a full-length human Endo G cDNA and comparison at the protein level of the human, bovine, and murine nucleases indicates a high degree of conservation except in the respective mitochondrial targeting signals. Endo G is ubiquitously expressed and the steady-state levels of its mRNA vary by a factor greater than seven between different tissues. The relationship between the mtDNA copy number and Endo G mRNA levels is not strictly proportional but tissues richer in mtDNA have higher amounts of the mRNA and vice versa.

Alternative Splicing↗
PubMed1997

A single cell complementation class is common to several cases of cytochrome c oxidase-defective Leigh's syndrome.

A generalized defect of complex IV (cytochrome C oxidase, COX) is frequently found in subacute necrotizing encephalomyelopathy (Leigh's syndrome), the most common mitochondrial disorder in infancy. We previously demonstrated the nuclear origin of the COX defect in one case, by fusing nuclear DNA-less cytoplasts derived from normal fibroblasts with mitochondrial DNA (mtDNA)-less transformant fibroblasts derived from a patient with COX-defective [COX(-)] Leigh's syndrome. The resulting cybrid line showed a specific and serve COX(-) phenotype. Conversely, in the present study, we demonstrated that a COX(+) phenotype could be restored in hybrids obtained by fusing COX(-) transformant fibroblasts of seven additional Leigh's syndrome patients with mtDNA-less, COX(-) tumor-derived rho degree cells. Both these results are explained by the presence of a mutation in a nuclear gene. In a second set of experiments, in order to demonstrate whether COX(-) Leigh's syndrome is due to a defect in the same gene, or in different genes, we tested several hybrids derived by fusing our original COX(-) cell line with each of the remaining seven cell lines. COX activity was evaluated in situ by histochemical techniques and in cell extracts by a spectrophotometric assay. No COX complementers were found among the resulting hybrid lines. This result demonstrates that all our cases were genetically homogeneous, and suggests that a major nuclear disease locus is associated with several, perhaps most, of the cases of infantile COX(-) Leigh's syndrome. This information should make it easier to identify the gene responsible.

Cell Fusion↗
PubMed1997

Identification of the gene encoding the human mitochondrial RNA polymerase (h-mtRPOL) by cyberscreening of the Expressed Sequence Tags database.

A gene cloning strategy based on the screening of the Expressed Sequence Tags database (dbEST) using sequences of mitochondrial housekeeping proteins of yeast was employed to identify the cDNA encoding the precursor of the human mitochondrial RNA polymerase (h-mtRPOL). The 3831 bp h-mtRPOL cDNA is located on chromosome 19p13.3 and encodes a protein of 1230 amino acid residues. The protein sequence shows significant homologies with sequences corresponding to mitochondrial RNA polymerases from lower eukaryotes, and to RNA polymerases from several bacteriophages. The mitochondrial RNA polymerase carries out the central activity of mitochondrial gene expression and, by providing the RNA primers for replication-initiation, is also implicated in the maintenance and propagation of the mitochondrial genome. Genes involved in the control of mtDNA replication and gene expression are attractive candidates for human disorders due to abnormalities of nucleo-mitochondrial intergenomic signalling. The availability of the h-mtRPOL cDNA will allow us to test its role in mitochondrial pathology. In addition, we propose the 'cyberscreening' of dbEST, based on yeast/human cross-species comparison, as a powerful, simple, rapid and inexpensive method, that may accelerate several-fold the molecular dissection of the human mitochondrial proteome.

Amino Acid Sequence↗
PubMed1997

Disorders of mitochondria and related metabolism.

Mitochondrial disorders are caused by mutations in either nuclear or mitochondrial genes involved in the synthesis of respiratory chain subunits or in their post-translational control. Molecular lesions of mitochondrial DNA are a frequent cause of defective oxidative phosphorylation. Although only one mutation of nuclear-encoded oxidative phosphorylation subunits has so far been reported in humans, numerous biochemically defined disorders are attributed to nuclear gene defects. The pathogenesis of these disorders has been investigated through a combination of different expertises, including keen clinical observation, classical biochemistry and muscle morphology, molecular and cellular biology, linkage analysis and population genetic studies.

Adult↗
PubMed1995

Searching for genes affecting the structural integrity of the mitochondrial genome.

Mendelian traits associated with qualitative or quantitative abnormalities of mtDNA are presumably caused by mutations in nucleus-encoded genes that deleteriously interact with the mitochondrial genome. Qualitative abnormalities of mtDNA are typically represented by pleioplasmic multiple mtDNA deletions, that are detected in stable tissues, including skeletal muscle, of patients affected by Autosomal Dominant Chronic Progressive External Ophthalmoplegia. Quantitative abnormalities are represented by tissue-specific depletion of mtDNA, associated with different clinical presentation in infancy or childhood. Linkage analysis and search for candidate genes are two complementary strategies aimed at identifying the genes responsible for these disorders.

Animals↗
PubMed1995

Chromosomal localization of mitochondrial transcription factor A (TCF6), single-stranded DNA-binding protein (SSBP), and endonuclease G (ENDOG), three human housekeeping genes involved in mitochondrial biogenesis.

By using a PCR-based screening of a somatic cell hybrid panel and FISH, we have assigned the loci of mitochondrial single-stranded DNA-binding protein (SSBP), mitochondrial transcription factor A (TCF6), and mitochondrial endonuclease G (ENDOG) genes to human chromosomes 7q34, 10q21, and 9q34.1, respectively. The products of these three genes are involved in fundamental aspects of mitochondrial biogenesis, such as replication and transcription of the mitochondrial genome. The chromosomal localization of these genes is important to testing whether the corresponding proteins may play a role in the etiopathogenesis of human disorders associated with qualitative or quantitative abnormalities of mitochondrial DNA.

Chromosome Mapping↗
PubMed1995

Sequence analysis of mitochondrial DNA in a new maternally inherited encephalomyopathy.

A heteroplasmic insertion of a 9-bp tandem repeat element was detected in the mitochondrial DNA of the maternal members of a large family. The mutation was contained within the non-coding region between the genes specifying subunit II of cytochrome c oxidase and tR-NA(Lys). The proband and most of his maternal relatives were affected by a late-onset mitochondrial encephalomyopathy of variable severity, characterized by a unique combination of symptoms. Extensive screening of a large series of DNA samples, collected from unrelated normal individuals as well as patients affected by different neurological disorders, consistently failed to detect the 9-bp insertion, with two exceptions: a patient suffering from a syndrome virtually identical to that described in our original family and a child affected by bilateral striatal necrosis, a disorder which has been attributed to impairment of mitochondrial oxidative phosphorylation. These considerations suggest that the 9-bp insertion is pathogenic and that the region affected by the mutation may play a previously unsuspected functional role in mtDNA gene expression.

Aged↗
PubMed1995

Early-onset encephalomyopathy associated with tissue-specific mitochondrial DNA depletion: a morphological, biochemical and molecular-genetic study.

A male infant, born from consanguineous parents, suffered from birth with a progressive neuromuscular disorder characterized by psychomotor delay, hypotonia, muscle weakness and wasting, deep-tendon areflexia and spastic posture. High levels of lactic acid in blood and cerebrospinal fluid suggested a mitochondrial respiratory chain defect. Muscle biopsy revealed ragged-red and cytochrome c oxidase-negative fibres, lipid accumulation and dystrophic changes. Multiple defects of respiratory complexes were detected in muscle homogenate, but cultured fibroblasts, myoblasts and myotubes were normal. Southern blot analysis showed markedly reduced levels of mitochondrial DNA (mtDNA) in muscle, while lymphocytes, fibroblasts and muscle precursor cells were normal. Neither depletion of mtDNA nor abnormalities of the respiratory complexes were observed in innervated muscle fibres cultured for as long as 4 months. No mutations were observed in two candidate nuclear genes, mtTFA and mtSSB, retro-transcribed, amplified and sequenced from the proband's mRNA. Sequence analysis of the mtDNA D-loop and of the origin of replication of the mtDNA light strand failed to identify potentially pathogenic mutations of these replicative elements in the proband's muscle mtDNA. Our findings indicate that mtDNA depletion is due to a nuclear encoded gene and suggest that the abnormality underlying defective mtDNA propagation must occur after muscle differentiation in vivo.

Age of Onset↗
PubMed1995

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 mitochondrial disorders. Faulty OXPHOS can be due to mutations in either nuclear or mitochondrial genes, that are involved in the synthesis of individual respiratory subunits or in their post-translational control. The most common mitochondrial disorder of infancy and childhood is Leigh's syndrome, a severe encephalopathy, often associated with a defect of cytochrome c oxidase (COX). In order to demonstrate which genome is primarily involved in COX-deficient (COX(-))-Leigh's syndrome, we generated two lines of transmitochondrial cybrids. The first was obtained by fusing nuclear DNA-less cytoplasts derived from normal fibroblasts, with mitochondrial DNA-less (rho degree) transformant fibroblasts derived from a patient with COX(-))-Leigh's syndrome. The second cybrid line was obtained by fusing rho degree cells derived from 143B.TK- human osteosarcoma cells, with cytoplasts derived from the same patient. The first cybrid line showed a specific and severe COX(-) phenotype, while in the second all the respiratory chain complexes, including COX, were normal. These results indicate that the COX defect in our patient is due to a mutation of a nuclear gene. The use of cybrids obtained from 'customized', patient-derived rho degree cells can have wide applications in the identification of respiratory chain defects originated by nuclear DNA-encoded mutations, and in the study of nuclear DNA-mitochondrial DNA interactions.

Cell Line↗
PubMed1995

Maternally inherited hearing loss, ataxia and myoclonus associated with a novel point mutation in mitochondrial tRNASer(UCN) gene.

We report on a new maternally-inherited syndrome characterized by a combination of sensorineural hearing loss, ataxia and myoclonus in a large kindred from Sicily. Hearing loss was the most widespread and sometimes the only symptom found in family members. Sequence analysis of the mitochondrial DNA regions encompassing the tRNA genes revealed the presence of a heteroplasmic insertion at nucleotide position 7472. The insertion adds a seventh cytosine to a six-cytosine run that is part of the mitochondrial tRNASer(UCN) gene. Conformational analysis showed that this mutation is likely to alter the structure of the T psi C loop in the tRNASer(UCN) clover leaf secondary structure. Moreover, the degree of heteroplasmy in blood and muscle was correlated with the clinical phenotype, and homoplasmic mutant hybrids showed decreased complex I activity, low oxygen consumption and high lactic acid output, indicating faulty oxidative phosphorylation. Finally, mutation was absent in 381 unrelated maternal lineages, suggesting specific segregation with the disease. We propose that the C7472 insertion-mutation is pathogenic, and etiologically related to hearing loss and other symptoms that define a novel maternally-inherited clinical entity.

Ataxia↗
PubMed1994

Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties.

The gene for the mature human mitochondrial single-stranded-DNA binding protein (HsmtSSB) has been transferred into a protein-overproducing vector and expressed in Escherichia coli. The protein was purified to homogeneity and its physicochemical properties were investigated. From sequence comparison, HsmtSSB shows some similarities to the N-terminal part of the single-stranded DNA-binding protein (SSB) from E. coli (EcoSSB). Hydrodynamic measurements show the protein to be tetrameric and give a sedimentation coefficient of 4.1 S corresponding to a C-terminally shortened EcoSSB. Electron-microscopic images of the free protein show a globular tetrahedral structure. Binding of poly(desoxythymidylic acid) [poly(dT)] leads to a reduction of the tryptophan fluorescence of the protein up to 96%. Fluorescence titrations with poly(dT) show apparent binding-site sizes of 50-70 nucleotides/tetramer between 0.05 M and 2 M NaCl. Binding to poly(dT) proceeds in a nearly diffusion-controlled reaction with an association-rate constant kass of 4 x 10(8) M-1s-1. The rate-limiting step is the formation of a transient complex where less than four binding sites on the protein are involved and the reshuffling of the protein on the linear matrix is fast. Electron microscopy of the complex with poly(dT) using negative staining shows a nearly random distribution of the protein between the individual poly(dT) strands. This leads to the conclusion that the binding cooperativity is low (omega < 150). The two tryptophans of HsmtSSB were replaced by threonine and tyrosine. The environment of both residues is influenced by nucleic acid binding with mutations of Trp68 strongly reducing the DNA-binding affinity of the protein.

Binding Sites↗