Biomedical subjects

Giorgio Lenaz

Publications and source records attributed to Giorgio Lenaz.

At least 19 recordsLinked to original sources

PubMed2006

Kinetics of integrated electron transfer in the mitochondrial respiratory chain: random collisions vs. solid state electron channeling.

Recent evidence, mainly based on native electrophoresis, has suggested that the mitochondrial respiratory chain is organized in the form of supercomplexes, due to the aggregation of the main respiratory chain enzymatic complexes. This evidence strongly contrasts the previously accepted model, the Random Diffusion Model, largely based on kinetic studies, stating that the complexes are randomly distributed in the lipid bilayer of the inner membrane and functionally connected by lateral diffusion of small redox molecules, i.e., coenzyme Q and cytochrome c. This review critically examines the experimental evidence, both structural and functional, pertaining to the two models and attempts to provide an updated view of the organization of the respiratory chain and of its kinetic consequences. The conclusion that structural respiratory assemblies exist is overwhelming, whereas the expected functional consequence of substrate channeling between the assembled enzymes is controversial. Examination of the available evidence suggests that, although the supercomplexes are structurally stable, their kinetic competence in substrate channeling is more labile and may depend on the system under investigation and the assay conditions.

Animals↗

PubMed2006

Defective oxidative phosphorylation in thyroid oncocytic carcinoma is associated with pathogenic mitochondrial DNA mutations affecting complexes I and III.

Oncocytic tumors are characterized by cells with an aberrant accumulation of mitochondria. To assess mitochondrial function in neoplastic oncocytic cells, we studied the thyroid oncocytic cell line XTC.UC1 and compared it with other thyroid non-oncocytic cell lines. Only XTC.UC1 cells were unable to survive in galactose, a condition forcing cells to rely solely on mitochondria for energy production. The rate of respiration and mitochondrial ATP synthesis driven by complex I substrates was severely reduced in XTC.UC1 cells. Furthermore, the enzymatic activity of complexes I and III was dramatically decreased in these cells compared with controls, in conjunction with a strongly enhanced production of reactive oxygen species. Osteosarcoma-derived transmitochondrial cell hybrids (cybrids) carrying XTC.UC1 mitochondrial DNA (mtDNA) were generated to discriminate whether the energetic failure depended on mitochondrial or nuclear DNA mutations. In galactose medium, XTC.UC1 cybrid clones showed reduced viability and ATP content, similarly to the parental XTC.UC1, clearly pointing to the existence of mtDNA alterations. Sequencing of XTC.UC1 mtDNA identified a frameshift mutation in ND1 and a nonconservative substitution in cytochrome b, two mutations with a clear pathogenic potential. In conclusion, this is the first demonstration that mitochondrial dysfunction of XTC.UC1 is due to a combined complex I/III defect associated with mtDNA mutations, as proven by the transfer of the defective energetic phenotype with the mitochondrial genome into the cybrids.

Adenoma, Oxyphilic↗

PubMed2006

Respiratory chain supercomplexes set the threshold for respiration defects in human mtDNA mutant cybrids.

Mitochondrial DNA (mtDNA) mutations cause heterogeneous disorders in humans. MtDNA exists in multiple copies per cell, and mutations need to accumulate beyond a critical threshold to cause disease, because coexisting wild-type mtDNA can complement the genetic defect. A better understanding of the molecular determinants of functional complementation among mtDNA molecules could help us shedding some light on the mechanisms modulating the phenotypic expression of mtDNA mutations in mitochondrial diseases. We studied mtDNA complementation in human cells by fusing two cell lines, one containing a homoplasmic mutation in a subunit of respiratory chain complex IV, COX I, and the other a distinct homoplasmic mutation in a subunit of complex III, cytochrome b. Upon cell fusion, respiration is recovered in hybrids cells, indicating that mitochondria fuse and exchange genetic and protein materials. Mitochondrial functional complementation occurs frequently, but with variable efficiency. We have investigated by native gel electrophoresis the molecular organization of the mitochondrial respiratory chain in complementing hybrid cells. We show that the recovery of mitochondrial respiration correlates with the presence of supramolecular structures (supercomplexes) containing complexes I, III and IV. We suggest that critical amounts of complexes III or IV are required in order for supercomplexes to form and provide mitochondrial functional complementation. From these findings, supercomplex assembly emerges as a necessary step for respiration, and its defect sets the threshold for respiratory impairment in mtDNA mutant cells.

Cell Line↗

PubMed2006

Mitochondrial Complex I: structural and functional aspects.

This review examines two aspects of the structure and function of mitochondrial Complex I (NADH Coenzyme Q oxidoreductase) that have become matter of recent debate. The supramolecular organization of Complex I and its structural relation with the remainder of the respiratory chain are uncertain. Although the random diffusion model [C.R. Hackenbrock, B. Chazotte, S.S. Gupte, The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport, J. Bioenerg. Biomembranes 18 (1986) 331-368] has been widely accepted, recent evidence suggests the presence of supramolecular aggregates. In particular, evidence for a Complex I-Complex III supercomplex stems from both structural and kinetic studies. Electron transfer in the supercomplex may occur by electron channelling through bound Coenzyme Q in equilibrium with the pool in the membrane lipids. The amount and nature of the lipids modify the aggregation state and there is evidence that lipid peroxidation induces supercomplex disaggregation. Another important aspect in Complex I is its capacity to reduce oxygen with formation of superoxide anion. The site of escape of the single electron is debated and either FMN, iron-sulphur clusters, and ubisemiquinone have been suggested. The finding in our laboratory that two classes of hydrophobic inhibitors have opposite effects on superoxide production favours an iron-sulphur cluster (presumably N2) is the direct oxygen reductant. The implications in human pathology of better knowledge on these aspects of Complex I structure and function are briefly discussed.

Animals↗

PubMed2006

Inefficient coupling between proton transport and ATP synthesis may be the pathogenic mechanism for NARP and Leigh syndrome resulting from the T8993G mutation in mtDNA.

Mutations in the ATP6 gene of mtDNA (mitochondrial DNA) have been shown to cause several different neurological disorders. The product of this gene is ATPase 6, an essential component of the F1F0-ATPase. In the present study we show that the function of the F1F0-ATPase is impaired in lymphocytes from ten individuals harbouring the mtDNA T8993G point mutation associated with NARP (neuropathy, ataxia and retinitis pigmentosa) and Leigh syndrome. We show that the impaired function of both the ATP synthase and the proton transport activity of the enzyme correlates with the amount of the mtDNA that is mutated, ranging from 13-94%. The fluorescent dye RH-123 (Rhodamine-123) was used as a probe to determine whether or not passive proton flux (i.e. from the intermembrane space to the matrix) is affected by the mutation. Under state 3 respiratory conditions, a slight difference in RH-123 fluorescence quenching kinetics was observed between mutant and control mitochondria that suggests a marginally lower F0 proton flux capacity in cells from patients. Moreover, independent of the cellular mutant load the specific inhibitor oligomycin induced a marked enhancement of the RH-123 quenching rate, which is associated with a block in proton conductivity through F0 [Linnett and Beechey (1979) Inhibitors of the ATP synthethase system. Methods Enzymol. 55, 472-518]. Overall, the results rule out the previously proposed proton block as the basis of the pathogenicity of the mtDNA T8993G mutation. Since the ATP synthesis rate was decreased by 70% in NARP patients compared with controls, we suggest that the T8993G mutation affects the coupling between proton translocation through F0 and ATP synthesis on F1. We discuss our findings in view of the current knowledge regarding the rotary mechanism of catalysis of the enzyme.

Adenosine Triphosphate↗

PubMed2006

Redox cycling of adrenaline and adrenochrome catalysed by mitochondrial Complex I.

Complex I in bovine heart submitochondrial particles catalyses the NADH-supported generation of superoxide anion; adrenaline is oxidised by superoxide to adrenochrome that, on its hand, is reduced by Complex I, thus establishing a redox cycle that amplifies the superoxide production. The routes in Complex I for superoxide formation and for adrenochrome reduction appear to be different, since they have a different sensitivity to Complex I inhibitors. The results are discussed in terms of current assays for superoxide detection and of pathologies linked to catecholamine oxidation.

Adrenochrome↗

PubMed2006

Mitochondrial activities of a cell line derived from thyroid Hürthle cell tumors.

OBJECTIVE: We conducted a study on the functional characteristics of mitochondria in an oxyphilic thyroid tumor cell line, which may provide useful clues about Hürthle cell tumors carcinogenesis. DESIGN: The functional study on thyroid tumors with cell oxyphilia (Hürthle cell tumors), characterized by mitochondrial hyperplasia, was carried out in XTC.UC1, and B-CPAP, an oxyphilic and nonoxyphilic thyroid tumor cell line, respectively. MAIN OUTCOME: XTC.UC1 cell line showed higher activity of mitochondrial respiratory complexes I and II and decreased activity of complex III. The increased activity of complex I was not matched by increased expression of complex I subunits. The XTC.UC1 cells relied mostly on oxidative phosphorylation for energy conservation, although their mitochondrial energetic function was less efficient when related to mitochondrial content of the cells. Finally, the oxyphilic cell line produced significantly higher amounts of reactive oxygen species (ROS) in comparison with B-CPAP cell line. CONCLUSION: The involvement of ROS in mitochondrial biogenesis and proliferation as well as in carcinogenesis and apoptosis indicate that differences in activity of respiratory chain components and their unbalance may be responsible for development of morphological and functional changes observed in thyroid tumors with cell oxyphilia.

Adenoma, Oxyphilic↗

PubMed2006

Hydrophobic alpha-helices 1 and 2 of herpes simplex virus gH interact with lipids, and their mimetic peptides enhance virus infection and fusion.

Entry of herpes simplex virus into cells occurs by fusion and requires four glycoproteins. gD serves as the receptor binding glycoprotein. Of the remaining glycoproteins, gH carries structural and functional elements typical of class 1 fusion glycoproteins, in particular alpha-helix 1 (alpha-H1), with properties of a candidate fusion peptide, and two heptad repeats. Here, we characterized alpha-H2 and compared it to alpha-H1. alpha-H2 (amino acids 513 to 531) is of lower hydrophobicity than alpha-H1. Its deletion or mutation decreased virus infection and cell fusion. Its replacement with heterologous fusion peptides did not rescue infection and cell fusion beyond the levels exhibited by the alpha-H2-deleted gH. This contrasts with alpha-H1, which cannot be deleted and can be functionally replaced with heterologous fusion peptides (T. Gianni et al., J. Virol. 79:2931-2940, 2005). Synthetic peptides mimicking alpha-H1 and alpha-H2 induced fusion of nude lipid vesicles. Importantly, they increased infection of herpes simplex virus, pseudorabies virus, bovine herpesvirus 1, and vesicular stomatitis virus. The alpha-H1 mimetic peptide was more effective than the alpha-H2 peptide. Consistent with the findings that gH carries membrane-interacting segments, a soluble form of gH, but not of gD or gB, partitioned with lipid vesicles. Current findings highlight that alpha-H2 is an important albeit nonessential region for virus entry and fusion. alpha-H1 and alpha-H2 share the ability to target the membrane lipids; they contribute to virus entry and fusion, possibly by destabilizing the membranes. However, alpha-H2 differs from alpha-H1 in that it is of lower hydrophobicity and cannot be replaced with heterologous fusion peptides.

Amino Acid Sequence↗

PubMed2005

Inhibition of glycerophosphate-dependent H2O2 generation in brown fat mitochondria by idebenone.

The established protective effect of coenzyme Q (CoQ) analogs is dependent on the location of reactive oxygen species (ROS) generation. One of these analogs--idebenone (hydroxydecyl-ubiquinone) is used as an antioxidative therapeutic drug. We tested its scavenging effect on the glycerophosphate (GP)-dependent ROS production as this enzyme was shown as a new site in the mitochondrial respiratory chain where ROS can be generated. We observed that idebenone inhibits both GP- and succinate-dependent ROS production. Idebenone and CoQ1 were found to be more efficient in the scavenging activity (IC50: 0.052 and 0.075 microM, respectively) than CoQ3 (IC50: 45.8 microM). Idebenone also inhibited ferricyanide (FeCN)-activated, GP-dependent ROS production. Our data thus extend previous findings on the scavenging effect of idebenone and show that it can also eliminate GP-dependent ROS generation.

Adipose Tissue, Brown↗

PubMed2005

Potential antitumor agents. 37. Synthesis and antitumor activity of guanylhydrazones from imidazo[2,1-b]thiazoles and from the new heterocyclic system thiazolo[2',3':2,3]imidazo[4,5-c]quinoline.

This paper reports synthesis and antitumor activity of new guanylhydrazones from imidazo[2,1-b]thiazoles and from the new heterocyclic system thiazolo[2',3':2,3]imidazo[4,5-c]quinoline. The compounds were tested as potential antitumor agents at the National Cancer Institute. The effect of the guanylhydrazone of 2-chloro-6-(2,5-dimethoxy-4-nitrophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde (41) was investigated, and it was found to be an inhibitor of Complex III of the mitochondrial respiratory chain and is able to induce apoptosis in the cell lines HT29 and HL60.

Animals↗

PubMed2005

Severe impairment of complex I-driven adenosine triphosphate synthesis in leber hereditary optic neuropathy cybrids.

BACKGROUND: Leber hereditary optic neuropathy (LHON) is a maternally inherited form of central vision loss associated with mitochondrial DNA point mutations that affect the ND subunits of complex I. OBJECTIVE: To elucidate the bioenergetic consequences of complex I dysfunction in LHON. DESIGN: The biochemical phenotypes of LHON mutations have been investigated using the transmitochondrial cytoplasmic hybrid (cybrid) cell model derived from the osteocarcoma parental cell line 143B.TK-. SETTING: Research laboratories at neuroscience and biochemistry departments at the University of Bologna, Scientific Institute "E. Medea," and University of College Medical School. PARTICIPANTS: Fibroblast cell lines were obtained from patients affected with LHON, as defined by the presence of 1 pathogenic mutation, and from healthy volunteers as controls to construct cybrid cell lines. MAIN OUTCOME MEASURES: Complex I (glutamate-malate)- and complex II (succinate)-dependent adenosine triphosphate (ATP) synthesis, their respective respiratory rates, and total cellular ATP content were investigated using digitonin permeabilized cybrid cells. Multiple cybrid cell lines were constructed, introducing into osteosarcoma-derived rho(0) cells either wild-type or LHON mutant mitochondria carrying each of the 3 common mutations at positions 11778/ND4, 3460/ND1, and 14484/ND6. RESULTS: All 3 LHON mutations impaired ATP synthesis and the respiratory control ratio driven by complex I substrates. In contrast, succinate-driven ATP synthesis, respiration rates, and respiratory control ratios were not affected. However, the defective ATP synthesis with complex I substrates did not result in reduced ATP cellular content, indicating a compensatory mechanism. CONCLUSIONS: The LHON pathogenic mutations profoundly impair complex I-dependent synthesis of ATP, providing a common biochemical feature that may play a major role in LHON pathogenesis. Stratification of the results by mutation suggests that the 11778/ND4 mutation may induce an uncoupling of cybrid respiration, whereas the other 2 mutations impair the oxygen consumption rate.

Adenosine Triphosphate↗

PubMed2005

Supercomplex organization of the mitochondrial respiratory chain and the role of the Coenzyme Q pool: pathophysiological implications.

In this review we examine early and recent evidence for an aggregated organization of the mitochondrial respiratory chain. Blue Native Electrophoresis suggests that in several types of mitochondria Complexes I, III and IV are aggregated as fixed supramolecular units having stoichiometric proportions of each individual complex. Kinetic evidence by flux control analysis agrees with this view, however the presence of Complex IV in bovine mitochondria cannot be demonstrated, presumably due to high levels of free Complex. Since most Coenzyme Q appears to be largely free in the lipid bilayer of the inner membrane, binding of Coenzyme Q molecules to the Complex I-III aggregate is forced by its dissociation equilibrium; furthermore free Coenzyme Q is required for succinate-supported respiration and reverse electron transfer. The advantage of the supercomplex organization is in a more efficient electron transfer by channelling of the redox intermediates and in the requirement of a supramolecular structure for the correct assembly of the individual complexes. Preliminary evidence suggests that dilution of the membrane proteins with extra phospholipids and lipid peroxidation may disrupt the supercomplex organization. This finding has pathophysiological implications, in view of the role of oxidative stress in the pathogenesis of many diseases.

Animals↗

PubMed2004

Biochemical analysis of respiratory function in cybrid cell lines harbouring mitochondrial DNA mutations.

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its rho(0) counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate levels were strictly and inversely correlated with rates of ATP synthesis and O2 consumption. These results show increased glycolysis and defective oxidative phosphorylation, irrespective of the type or site of the point mutation or deletion in the mitochondrial genome. The different biochemical consequences of the T8993C mutation suggest a uniquely different pathogenic mechanism for this mutation. However, the distinct clinical features associated with some of these mutations still remain to be elucidated.

Adenosine Triphosphate↗

PubMed2004

Effects of new ubiquinone-imidazo[2,1-b]thiazoles on mitochondrial complex I (NADH-ubiquinone reductase) and on mitochondrial permeability transition pore.

In this work we describe the synthesis of a series of imidazo[2,1-b]thiazoles and 2,3-dihydroimidazo[2,1-b]thiazoles connected by means of a methylene bridge to CoQ(0). These compounds were tested as specific inhibitors of the NADH:ubiquinone reductase activity in mitochondrial membranes. The imidazothiazole system when bound to the quinone ring in place of the isoprenoid lateral side chain, may increase the inhibitory effect (with an IC(50) for NADH-Q(1) activity ranging between 0.25 and 0.96 microM) whereas the benzoquinone moiety seems to lose the capability to accept electrons from complex I as indicated by very low maximal velocity elicited by the compounds tested. Moreover the low rotenone sensitivity for almost all of these compounds suggests that they are only partially able to interact with the physiological ubiquinone-reduction site. The compounds were investigated for the capability of increasing the permeability transition of the inner mitochondrial membrane in isolated mitochondria. Unlike CoQ(0), which is considered a mitochondrial membrane permeability transition inhibitor, the new compounds were inducers.

Animals↗

PubMed2004

Heterologous mitochondrial DNA recombination in human cells.

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

Blotting, Southern↗

PubMed2004

Bioenergetics of mitochondrial diseases associated with mtDNA mutations.

This mini-review summarizes our present view of the biochemical alterations associated with mitochondrial DNA (mtDNA) point mutations. Mitochondrial cytopathies caused by mutations of mtDNA are well-known genetic and clinical entities, but the biochemical pathogenic mechanisms are often obscure. Leber's hereditary optic neuropathy (LHON) is due to three main mutations in genes for complex I subunits. Even if the catalytic activity of complex I is maintained except in cells carrying the 3460/ND1 mutation, in all cases there is a change in sensitivity to complex I inhibitors and an impairment of mitochondrial respiration, eliciting the possibility of generation of reactive oxygen species (ROS) by the complex. Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa (NARP), is due to a mutation in the ATPase-6 gene. In NARP patients ATP synthesis is strongly depressed to an extent proportional to the mutation load; nevertheless, ATP hydrolysis and ATP-driven proton translocation are not affected. It is suggested that the NARP mutation affects the ability of the enzyme to couple proton transport to ATP synthesis. A point mutation in subunit III of cytochrome c oxidase is accompanied by a syndrome resembling MELAS: however, no major biochemical defect is found, if we except an enhanced production of ROS. The mechanism of such enhancement is at present unknown. In this review, we draw attention to a few examples in which the overproduction of ROS might represent a common step in the induction of clinical phenotypes and/or in the progression of several human pathologies associated with mtDNA point mutations.

Adenosine Triphosphatases↗

PubMed2004

Bioenergetics shapes cellular death pathways in Leber's hereditary optic neuropathy: a model of mitochondrial neurodegeneration.

Leber's hereditary optic neuropathy (LHON) was the first maternally inherited disease to be associated with point mutations in mitochondrial DNA and is now considered the most prevalent mitochondrial disorder. The pathology is characterized by selective loss of ganglion cells in the retina leading to central vision loss and optic atrophy, prevalently in young males. The pathogenic mtDNA point mutations for LHON affect complex I with the double effect of lowering the ATP synthesis driven by complex I substrates and increasing oxidative stress chronically. In this review, we first consider the biochemical changes associated with the proton-translocating NADH-quinone oxidoreductase of mitochondria in cybrid cells carrying the most common LHON mutations. However, the LHON cybrid bioenergetic dysfunction is essentially compensated under normal conditions, i.e. in glucose medium, but is unrevealed by stressful conditions such as growing cybrids in glucose free/galactose medium, which forces cells to rely only on respiratory chain for ATP synthesis. In fact, the second part of this review deals with the investigation of LHON cybrid death pathway in galactose medium. The parallel marked changes in antioxidant enzymes, during the time-course of galactose experiments, also reveal a relevant role played by oxidative stress. The LHON cybrid model sheds light on the complex interplay amongst the different levels of biochemical consequences deriving from complex I mutations in determining neurodegeneration in LHON, and suggests an unsuspected role of bioenergetics in shaping cell death pathways.

Animals↗

PubMed2004

The mitochondrial respiratory chain is partially organized in a supercomplex assembly: kinetic evidence using flux control analysis.

The model of the respiratory chain in which the enzyme complexes are independently embedded in the lipid bilayer of the inner mitochondrial membrane and connected by randomly diffusing coenzyme Q and cytochrome c is mostly favored. However, multicomplex units can be isolated from mammalian mitochondria, suggesting a model based on direct electron channeling between complexes. Kinetic testing using metabolic flux control analysis can discriminate between the two models: the former model implies that each enzyme may be rate-controlling to a different extent, whereas in the latter, the whole metabolic pathway would behave as a single supercomplex and inhibition of any one of its components would elicit the same flux control. In particular, in the absence of other components of the oxidative phosphorylation apparatus (i.e. ATP synthase, membrane potential, carriers), the existence of a supercomplex would elicit a flux control coefficient near unity for each respiratory complex, and the sum of all coefficients would be well above unity. Using bovine heart mitochondria and submitochondrial particles devoid of substrate permeability barriers, we investigated the flux control coefficients of the complexes involved in aerobic NADH oxidation (I, III, IV) and in succinate oxidation (II, III, IV). Both Complexes I and III were found to be highly rate-controlling over NADH oxidation, a strong kinetic evidence suggesting the existence of functionally relevant association between the two complexes, whereas Complex IV appears randomly distributed. Moreover, we show that Complex II is fully rate-limiting for succinate oxidation, clearly indicating the absence of substrate channeling toward Complexes III and IV.

Adenosine Triphosphate↗