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Biomedical subjects

Giuseppe Attardi

Publications and source records attributed to Giuseppe Attardi.

15 recordsLinked to original sources
PubMed2006

Identification of a novel mitochondrial complex containing mitofusin 2 and stomatin-like protein 2.

A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an approximately 42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062-8071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a "ladder" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology.

Blood Proteins↗
PubMed2006

Hypersensitivity to oxygen and shortened lifespan in a Drosophila mitochondrial complex II mutant.

Oxidative stress is implicated as a major cause of aging and age-related diseases, such as Parkinson's and Alzheimer's, as well as ischemia-reperfusion injury in stroke. The mitochondrial electron transport chain is the principal source of reactive oxygen species within cells. Despite considerable medical interest, the molecular mechanisms that regulate reactive oxygen species formation within the mitochondrion remain poorly understood. Here, we report the isolation and characterization of a Drosophila mutant with a defect in subunit b of succinate dehydrogenase (SDH; mitochondrial complex II). The sdhB mutant is hypersensitive to oxygen and displays hallmarks of a progeroid syndrome, including early-onset mortality and age-related behavioral decay. Pathological analysis of the flight muscle, which is amongst the most highly energetic tissues in the animal kingdom, reveals structural abnormalities in the mitochondria. Biochemical analysis shows that, in the mutant, there is a complex II-specific respiratory defect and impaired complex II-mediated electron transport, although the other respiratory complexes remain functionally intact. The complex II defect is associated with an increased level of mitochondrial hydrogen peroxide production, suggesting a possible mechanism for the observed sensitivity to elevated oxygen concentration and the decreased lifespan of the mutant fly.

Aging↗
PubMed2006

Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology.

Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase gamma. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.

Animals↗
PubMed2005

Termination factor-mediated DNA loop between termination and initiation sites drives mitochondrial rRNA synthesis.

The human mitochondrial transcription termination factor mTERF plays a central role in the control of heavy-strand rDNA transcription by promoting initiation, besides termination, of this transcription. However, until now, the mechanism underlying this stimulation of transcription by mTERF was not understood. In the present work, addition of mTERF to a HeLa cell mitochondrial lysate-based reaction mixture containing an artificial rDNA template did indeed specifically stimulate rDNA transcription. This stimulation required that mTERF be simultaneously bound to the rDNA transcription termination and initiation sites in the same molecule, thus forming a loop. Most significantly, a double binding of mTERF to the rDNA molecule, with resulting loop formation, was also shown in vivo. These results strongly suggest that, to satisfy the need for high rate of rDNA transcription, human mitochondrial rRNA synthesis involves mTERF-mediated rDNA looping that promotes recycling of the transcription machinery.

Cell-Free System↗
PubMed2004

Discovery of a major D-loop replication origin reveals two modes of human mtDNA synthesis.

Mammalian mitochondrial DNA (mtDNA) replication has long been considered to occur by asymmetric synthesis of the two strands, starting at the multiple origins of the strand-displacement loop (D-loop). We report the discovery of a major replication origin at position 57 in the D-loop of several human cell lines (HeLa, A549, and 143B.TK-) and immortalized lymphocytes. The nascent chains starting at this origin, in contrast to those initiated at the previously described origins, do not terminate prematurely at the 3' end of the D-loop but proceed well beyond this control point, behaving as "true" replicating strands. This origin is mainly responsible for mtDNA maintenance under steady-state conditions, whereas mtDNA synthesis from the formerly identified D-loop origins may be more important for recovery after mtDNA depletion and for accelerating mtDNA replication in response to physiological demands.

Cell Line↗
PubMed2004

Nuclear control of cloverleaf structure of human mitochondrial tRNA(Lys).

The evolutionary loss in eukaryotic cells of mitochondrial (mt) tRNA genes and of tRNA structural information in the surviving genes has led to the appearance of mt-tRNAs with highly unusual structural features. One such mt-tRNA is the human mt-tRNALys, which relies on post-transcriptional base modification to achieve correct three-dimensional structure. It has been shown that the in vitro transcript of human mt-tRNALys adopts a particular, non-cloverleaf structure when devoid of modified bases, while the native, fully modified tRNA shows the expected cloverleaf structure. Furthermore, a methyl group at position A9-N1, introduced chemically in an otherwise unmodified mt-tRNALys transcript, was found to induce a stable cloverleaf conformation, raising the question of how the specific methyltransferase recognizes the unmodified transcript. In order to shed light on this unusual case of tRNA maturation, the tRNA modification enzymes contained in protein extracts from either highly purified HeLa cell mitochondria or HeLa cell cytosol were first identified and compared, and then used to analyze the mt-tRNALys. An initial screening for modification activities, using as substrates unmodified in vitro transcripts of tRNA genes with well characterized structures, namely yeast cytosolic tRNAPhe, human cytosolic tRNA3Lys, and human mt-tRNAIle, revealed the presence of nine and 11 modification activities in the mitochondrial and cytosolic protein extracts, respectively, the mitochondrial extract including a tRNA (adenine-9,N1)-methyltransferase activity. The comparison of the level and kinetics of A9-N1 methylation and other secondary modifications in the unmodified, misfolded mt-tRNALys and in a cloverleaf-shaped structural mutant, engineered to adopt the tRNALys cloverleaf structure without post-transcriptional modifications, suggested strongly that the methylation of A9-N1 in tRNALys proceeds via a cloverleaf-shaped intermediate. Therefore, it is proposed that this intermediate is present in the in vitro transcript as part of a dynamic equilibrium, and that the mitochondrial protein extract contains an activity that stabilizes, by secondary modification, such a transient cloverleaf-shaped intermediate. Thus, countering the evolutionary loss of structural information in mt-tRNA genes, the mt-tRNA structure is maintained by a modification enzyme encoded in nuclear DNA.

Cytosol↗
PubMed2004

A monomer-to-trimer transition of the human mitochondrial transcription termination factor (mTERF) is associated with a loss of in vitro activity.

The human mitochondrial transcription termination factor (mTERF) is a nuclear-encoded 39-kDa protein that recognizes a mtDNA segment within the mitochondrial tRNA(Leu(UUR)) gene immediately adjacent to and downstream of the 16 S rRNA gene. Binding of mTERF to this site promotes termination of rDNA transcription. Despite the fact that mTERF binds DNA as a monomer, the presence in its sequence of three leucine-zipper motifs suggested the possibility of mTERF establishing intermolecular interactions with proteins of the same or different type. When a mitochondrial lysate from HeLa cells was submitted to gel filtration chromatography, mTERF was eluted in two peaks, as detected by immunoblotting. The first peak, which varied in proportion between 30 and 50%, appeared at the position expected from the molecular mass of the monomer (41 +/- 2 kDa), and the gel filtration fractions that contained it exhibited DNA binding activity. Most interestingly, the material in this peak had a strong stimulating activity on in vitro transcription of the mitochondrial rDNA. The second peak eluted at a position corresponding to an estimated molecular mass of 111 +/- 5 kDa. No mTERF DNA binding activity could be detected in the corresponding gel filtration fractions. Therefore, we propose that mTERF exists in mitochondria in two forms, an active monomer and an inactive large size complex. The estimated molecular weight of this complex and the fact that purified mTERF can be eluted from a gel filtration column as a complex of the same molecular weight strongly suggest that this inactive complex is a homotrimer of mTERF.

Amino Acid Motifs↗
PubMed2004

Genetic and functional analysis of mitochondrial DNA-encoded complex I genes.

Mammalian mitochondrial NADH dehydrogenase (complex I) is a multimeric complex consisting of at least 45 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). The function of these subunits is largely unknown. We have established an efficient method to isolate and characterize cells carrying mutations in various mtDNA-encoded complex I genes. With this method, 15 mouse cell lines with deficiencies in complex I-dependent respiration were obtained, and two near-homoplasmic mutations in mouse ND5 and ND6 genes were isolated. Furthermore, by generating a series of cell lines with the same nuclear background but different content of an mtDNA nonsense mutation, we analyzed the genetic and functional thresholds in mouse mitochondria. We found that in wild-type cells, about 40% of ND5 mRNA is in excess of that required to support a normal rate of ND5 subunit synthesis. However, there is no indication of compensatory upsurge in either transcription or translation with the increase in the proportion of mutant ND5 genes. Interestingly, the highest ND5 protein synthesis rate was just sufficient to support the maximum complex I-dependent respiration rate, suggesting a tight regulation at the translational level. In another line of research, we showed that the mitochondrial NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1), although consisting of a single subunit, can completely restore respiratory NADH dehydrogenase activity in mutant human cells that lack the essential mtDNA-encoded subunit ND4. In particular, in these transfected cells, the yeast enzyme becomes integrated into the human respiratory chain and fully restores the capacity of the cells to grow in galactose medium.

Animals↗
PubMed2003

Marked aging-related decline in efficiency of oxidative phosphorylation in human skin fibroblasts.

An extensive analysis has been carried out of mitochondrial biochemical and bioenergetic properties of fibroblasts, mostly skin-derived, from a large group of subjects ranging in age between 20 wk fetal and 103 yr. A striking age-related change observed in a fundamental process underlying mitochondrial biogenesis and function was the very significant decrease in rate of mitochondrial protein synthesis in individuals above 40 yr. The analysis of endogenous respiration rate revealed a significant decrease in the age range from 40 to 90 yr and a tendency to uncoupling in the samples from subjects above 60 yr. A surprising finding was the occurrence of a subgroup of individuals >or=90 yr old whose skin fibroblasts exhibited an exceptionally high respiration rate. This high rate was not due to respiration uncoupling, rather pointing to a compensatory phenomenon, not involving an increase in mtDNA content, in the corresponding skin fibroblast populations, or, possibly, to a selection of a different cell type secondary to more extensive dermal atrophy. The most important aging-related phenotypic effects observed were those that affected the cell oxidative phosphorylation (OX-PHOS) capacity. These were, in particular, the very significant reduction in the ratio of uncoupled to oligomycin-inhibited endogenous respiration observed in intact fibroblasts, which pointed to a decrease with donor's age in the control of respiration by the mitochondrial membrane potential, the very significant decrease in efficiency of OX-PHOS, as determined by novel in situ measurements of P:O ratios, and, consistent with these results, the very significant reduction in the respiratory control ratios. These findings clearly pointed to a dramatic mitochondrial dysfunction, which would lead to a decrease in ATP synthesis rate, with the observed decline in mitochondrial protein synthesis rate being a likely contributing factor. These observations have important implications for understanding the biology of aging, as well as the pathogenesis of aging-related degenerative diseases.

Adolescent↗
PubMed2003

MtDNA mutations in aging and apoptosis.

There is considerable evidence that the oxidative phosphorylation capacity of human mitochondria declines in various tissues with aging. However, the genetic basis of this phenomenon has not yet been clarified. The occurrence of large deletions in mtDNA from brain, skeletal, and heart muscles and other tissues of old subjects at relatively low levels has been well documented. We discuss their possible functional relevance for the aging processes. On the contrary, until very recently, only inconclusive and often discordant evidence was available for the accumulation of mtDNA point mutations in old individuals. In the past few years, however, an aging-dependent large accumulation of mtDNA point mutations has been demonstrated in the majority of individuals above a certain age. These mutations occur in the mtDNA main control region at critical sites for mtDNA replication in fibroblasts and skeletal muscles. The extraordinary tissue specificity and nucleotide selectivity of these mutations strongly support the idea of their being functionally relevant. Evidence in agreement with this conclusion has been provided by the very recent observation that an mtDNA mutation occurring in blood leukocytes near an origin of replication, which causes a remodeling of this origin, occurs at a strikingly higher frequency in centenarians and monozygotic and dizygotic twins than in the control populations, strongly pointing to its survival value. The present article reviews another area of active research and discussion, namely, the role of pathogenic mtDNA mutations in causing programmed cell death. The available evidence has clearly shown that mtDNA and respiration are not essential for the process of apoptosis. However, the limited and sometimes contradictory data indicate that the absence or impaired function of mtDNA can influence the rate of this process, most probably by regulating the production of reactive oxygen species or the lack thereof.

Aging↗
PubMed2003

Strikingly higher frequency in centenarians and twins of mtDNA mutation causing remodeling of replication origin in leukocytes.

The presence of a genetic component in longevity is well known. Here, the association of a mtDNA mutation with a prolonged life span in humans was investigated. Large-scale screening of the mtDNA main control region in leukocytes from subjects of an Italian population revealed a homoplasmic C150T transition near an origin of heavy mtDNA-strand synthesis in approximately 17% of 52 subjects 99-106 years old, but, in contrast, in only 3.4% of 117 younger individuals (P = 0.0035). Evidence was obtained for the contribution of somatic events, under probable nuclear genetic control, to the striking selective accumulation of the mutation in centenarians. In another study, among leukocyte mtDNA samples from 20 monozygotic and 18 dizygotic twins, 60-75 years old, 30% (P = 0.0007) and 22% (P = 0.011), respectively, of the individuals involved exhibited the homoplasmic C150T mutation. In a different system, i.e., in five human fibroblast longitudinal studies, convincing evidence for the aging-related somatic expansion of the C150T mutation, up to homoplasmy, was obtained. Most significantly, 5' end analysis of nascent heavy mtDNA strands consistently revealed a new replication origin at position 149, substituting for that at 151, only in C150T mutation-carrying samples of fibroblasts or immortalized lymphocytes. Considering the aging-related health risks that the centenarians have survived and the developmental risks of twin gestations, it is proposed that selection for a remodeled replication origin, inherited or somatically acquired, provides a survival advantage and underlies the observed high incidence of the C150T mutation in centenarians and twins.

Adolescent↗
PubMed2002

Mitochondrial outer membrane permeability change and hypersensitivity to digitonin early in staurosporine-induced apoptosis.

We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK(-) cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK(-) osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK(-) cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.

Apoptosis↗