Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from Complex I

Evidence accumulated in the last ten years has demonstrated that a large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes or respirasomes. Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. Following this line of thought we have decided to directly investigate ROS production by Complex I under conditions in which the complex is arranged as a component of the supercomplex I1III2 or it is dissociated as an individual enzyme. The study has been addressed both in bovine heart mitochondrial membranes and in reconstituted proteoliposomes composed of complexes I and III in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. The results of this investigation provide experimental evidence that the production of ROS is strongly increased in either model; supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I . This is the first demonstration that dissociation of the supercomplex I1III2 in the mitochondrial membrane is a cause of oxidative stress from Complex I. Previous work in our laboratory demonstrated that lipid peroxidation can dissociate the supramolecular assemblies; thus, here we confirm that preliminary conclusion that primary causes of oxidative stress may perpetuate reactive oxygen species (ROS) generation by a vicious circle involving supercomplex dissociation as a major determinant.

Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

Studio della subunità epsilon della DNA polimerasi III di Escherichia coli: stabilità e interazione con la subunità polimerasica

Faithful replication of DNA from one generation to the next is crucial for long-term species survival. Genomic integrity in prokaryotes, archaea and eukaryotes is dependent on efficient and accurate catalysis by multiple DNA polymerases. Escherichia coli possesses five known DNA polymerases (Pol). DNA polymerase III holoenzyme is the major replicative polymerase of the Escherichia coli chromosome (Kornberg, 1982). This enzyme contains two Pol III cores that are held together by a t dimer (Studwell-Vaughan and O’Donnell, 1991). The core is composed of three different proteins named α-, ε- and θ-subunit. The α-subunit, encoded by dnaE, contains the catalytic site for DNA polymerisation (Maki and Kornberg, 1985), the ε-subunit, encoded by dnaQ, contains the 3′→5′ proofreading exonuclease (Scheuermann, et al., 1983) and the θ-subunit, encoded by hole, that has no catalytic activity (Studwell-Vaughan, and O'Donnell, 1983). The three-subunit α–ε–θ DNA pol III complex is the minimal active polymerase form purified from the DNA pol III holoenzyme complex; these three polypeptides are tightly associated in the core (McHenry and Crow, 1979) Despite a wealth of data concerning the properties of DNA polymerase III in vitro, little information is available on the assembly in vivo of this complex enzyme. In this study it is shown that the C-terminal region of the proofreading subunit is labile and that the ClpP protease and the molecular chaperones GroL and DnaK control the overall concentration in vivo of ε. Two α-helices (comprising the residues E311-M335 and G339-D353, respectively) of the N-terminal region of the polymerase subunit were shown to be essential for the binding to ε. These informations could be utilized to produce a conditional mutator strain in which proofreading activity would be titrated by a a variant that can only bind e and that is polymerase-deficient. In this way the replication of DNA made by DNA Pol-III holoenzyme would accordingly become error-prone.

Thermochronologic and geodynamic evolution of the Pontides: Sakarya terrane (Karakaya Complex) and Istanbul terrane, Turkey.

An integrated array of analytical methods -including clay mineralogy, vitrinite reflectance, Raman spectroscopy on carbonaceous material, and apatite fission-track analysis- was employed to constrain the thermal and thermochronological evolution of selected portions of the Pontides of northern Turkey. (1) A multimethod investigation was applied for the first time to characterise the thermal history of the Karakaya Complex, a Permo-Triassic subduction-accretion complex cropping out throughout the Sakarya Zone. The results indicate two different thermal regimes: the Lower Karakaya Complex (Nilüfer Unit) -mostly made of metabasite and marble- suffered peak temperatures of 300-500°C (greenschist facies); the Upper Karakaya Complex (Hodul and the Orhanlar Units) –mostly made of greywacke and arkose- yielded heterogeneous peak temperatures (125-376°C), possibly the result of different degree of involvement of the units in the complex dynamic processes of the accretionary wedge. Contrary to common belief, the results of this study indicate that the entire Karakaya Complex suffered metamorphic conditions. Moreover, a good degree of correlation among the results of these methods demonstrate that Raman spectroscopy on carbonaceous material can be applied successfully to temperature ranges of 200-330°C, thus extending the application of this method from higher grade metamorphic contexts to lower grade metamorphic conditions. (2) Apatite fission-track analysis was applied to the Sakarya and the İstanbul Zones in order to constrain the exhumation history and timing of amalgamation of these two exotic terranes. AFT ages from the İstanbul and Sakarya terranes recorded three distinct episodes of exhumation related to the complex tectonic evolution of the Pontides. (i) Paleocene - early Eocene ages (62.3-50.3 Ma) reflect the closure of the İzmir-Ankara ocean and the ensuing collision between the Sakarya terrane and the Anatolide-Tauride Block. (ii) Late Eocene - earliest Oligocene (43.5-32.3 Ma) ages reflect renewed tectonic activity along the İzmir-Ankara. (iii) Late Oligocene- Early Miocene ages reflect the onset and development of the northern Aegean extension. The consistency of AFT ages, both north and south of the tectonic contact between the İstanbul and Sakarya terranes, suggest that such terranes were amalgamated in pre-Cenozoic times. (3) Fission-track analysis was also applied to rock samples from the Marmara region, in an attempt to constrain the inception and development of the North Anatolian Fault system in the region. The results agree with those from the central Pontides. The youngest AFT ages (Late Oligocene - early Miocene) were recorded in the western portion of the Marmara Sea region and reflect the onset and development of northern Aegean extension. Fission-track data from the eastern Marmara Sea region indicate rapid Early Eocene exhumation induced by the development of the İzmir-Ankara orogenic wedge. Thermochronological data along the trace of the Ganos Fault –a segment of the North Anatolian Fault system- indicate the presence of a tectonic discontinuity active by Late Oligocene time, i.e. well before the arrival of the North Anatolian Fault system in the area. The integration of thermochronologic data with preexisting structural data point to the existence of a system of major E-W-trending structural discontinuities active at least from the Late Oligocene. In the Early Pliocene, inception of the present-day North Anatolian Fault system in the Marmara region occurred by reactivation of these older tectonic structures. 


Funzioni della subunità θ e del dominio PHP della subunità α nel core catalitico della DNA polimerasi III di Escherichia coli

Il core catalitico della DNA polimerasi III, composto dalle tre subunità α, ε e θ, è il complesso minimo responsabile della replicazione del DNA cromosomiale in Escherichia coli. Nell'oloenzima, α ed ε possiedono rispettivamente un'attività 5'-3' polimerasica ed un'attività 3'-5' esonucleasica, mentre θ non ha funzioni enzimatiche. Il presente studio si è concentrato sulle regioni del core che interagiscono direttamente con ε, ovvero θ (interagente all'estremità N-terminale di ε) e il dominio PHP di α (interagente all'estremità C-terminale di ε), delle quali non è stato sinora identificato il ruolo. Al fine di assegnare loro una funzione sono state seguite tre linee di ricerca parallele. Innanzitutto il ruolo di θ è stato studiato utilizzando approcci ex-vivo ed in vivo. I risultati presentati in questo studio mostrano che θ incrementa significativamente la stabilità della subunità ε, intrinsecamente labile. Durante gli esperimenti condotti è stata anche identificata una nuova forma dimerica di ε. Per quanto la funzione del dimero non sia definita, si è dimostrato che esso è attivamente dissociato da θ, che potrebbe quindi fungere da suo regolatore. Inoltre, è stato ritrovato e caratterizzato il primo fenotipo di θ associato alla crescita. Per quanto concerne il dominio PHP, si è dimostrato che esso possiede un'attività pirofosfatasica utilizzando un nuovo saggio, progettato per seguire le cinetiche di reazione catalizzate da enzimi rilascianti fosfato o pirofosfato. L'idrolisi del pirofosfato catalizzata dal PHP è stata dimostrata in grado di sostenere l'attività polimerasica di α in vitro, il che suggerisce il suo possibile ruolo in vivo durante la replicazione del DNA. Infine, è stata messa a punto una nuova procedura per la coespressione e purificazione del complesso α-ε-θ

Structural and kinetic characterization of DNA polymerases I and III from Escherichia coli

DNA elongation is performed by Pol III α subunit in E. coli, stimulated by the association with ε and θ subunits. These three subunits define the DNA Pol III catalytic core. There is controversy about the DNA Pol III assembly for the simultaneous control of lagging and leading strands replication, since some Authors propose a dimeric model with two cores, whereas others have assembled in vitro a trimeric DNA Pol III with a third catalytic core, which increases the efficiency of DNA replication. Moreover, the function of the PHP domain, located at the N-terminus of α subunit, is still unknown. Previous studies hypothesized a possible pyrophosphatase activity, not confirmed yet. The present Thesis highlights by the first time the production in vivo of a trimeric E. coli DNA Pol III by co-expressing α, τ, ε and θ subunits. This trimeric complex has been enzymatically characterized and a molecular model has been proposed, with 2 α subunits sustaining the lagging-strand replication whereas the third core replicates the leading strand. In addition, the pyrophosphatase activity of the PHP domain has been confirmed. This activity involves, at least, the H12 and the D19 residues, whereas the D201 regulates phosphate release. On the other hand, an artificial polymerase (HoLaMa), designed by deleting the exonuclease domain of Klenow Fragment, has been expressed, purified and characterized for a better understanding of bacterial polymerases mechanism. The absence of exonuclease domain impaired enzyme processivity, since this domain is involved in DNA binding. Finally, Klenow enzyme, HoLaMa, α subunit and DNA Pol III αεθ have been characterized at the single-molecule level by FRET analysis, combining ALEX and TIRF microscopy. Fluorescently-labeled DNA molecules were immobilized, and changes in FRET efficiency enabled us to study polymerase binding and DNA polymerization.