Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
Respiratory complex III (CIII) is the central complex of the respiratory chain. In its native for... more Respiratory complex III (CIII) is the central complex of the respiratory chain. In its native form, CIII is dimeric, and is closely associated in varying proportions with CI and CIV to form supramolecular structures, called supercomplexes [1]. The occurrence of such supercomplexes as structural and functional entities has been recently documented [1,2]. However, the physiological implications of such specific supramolecular interactions are not yet fully understood. Mutations in cytochrome b, which is the only CIII subunit encoded by the mitochondrial genome, are among the least common abnormalities identified to date in humans [3]. Recent studies on the bacterial enzyme proposed that specific amino acids at the Q o site of CIII might provide protective mechanisms against oxidative damage. Interestingly, the conserved Tyr residue at position 302 of Rhodobacter capsulatus cytochrome b is critical for this process [4]. The same mutation at position 278 of human cytochrome b (p.Y278C, m.15579ANG) has also been encountered in a patient with severe exercise intolerance and multisystem disorder [5]. Here we have utilized cybrids bearing the homoplasmic p.Y278C mutation to dissect the biochemical alterations in CIII activity, ROS production and supercomplexes assembly/stability. Despite a dramatic reduction in CIII activity, and in CIII-driven ATP synthesis, the CI and I + III activities were less affected, and the rate of ATP synthesis driven by CI or CII substrates was only partially reduced. Accordingly, mutated cybrids maintained the mitochondrial potential in the presence of oligomycin. Remarkably, the p.Y278C mutation enhanced superoxide production and perturbed glutathione homeostasis. Finally, examination of respiratory supercomplexes revealed that dimeric CIII and III 2 IV 1 were markedly decreased, whereas supercomplexes I 1 III 2 IV n increased. These findings suggest that the deleterious effects of cytochrome b p.Y278C mutation are mitigated when CIII is assembled into the supercomplexes I 1 III 2 IV n and underline the importance of supramolecular interactions between respiratory complexes on disease manifestations associated with this mutation.
OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plu... more OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plus, pathologies for which there is no established cure. We used a ‘drug repurposing’ approach to identify FDA-approved molecules able to rescue the mitochondrial dysfunctions induced by OPA1 mutations. We screened two different chemical libraries by using two yeast strains carrying the mgm1I322M and the chim3P646L mutations, identifying 26 drugs able to rescue their oxidative growth phenotype. Six of them, able to reduce the mitochondrial DNA instability in yeast, have been then tested in Opa1 deleted mouse embryonic fibroblasts expressing the human OPA1 isoform 1 bearing the R445H and D603H mutations. Some of these molecules were able to ameliorate the energetic functions and/or the mitochondrial network morphology, depending on the type of OPA1 mutation. The final validation has been performed in patients’ fibroblasts, allowing to select the most effective molecules. Our current results...
Redox Proteins in Supercomplexes and Signalosomes, 2015
In eukaryotic cells, mitochondria play the fundamental role of ATP production during the process ... more In eukaryotic cells, mitochondria play the fundamental role of ATP production during the process of oxidative phosphorylation (OXPHOS). However, these cytosolic organelles also have several other important physiological functions, including sugar and fatty acid catabo- lism, amino acid metabolism, buffering of the cytosolic calcium concentration (Rizzuto et\\ua0al., 2012), regulation and execution of different types of cell death (Galluzzi et\\ua0al., 2012) and arrangement of adaptive responses to perturbations of intracellular homeostasis (Liesa and Shirihai, 2013). Furthermore, mitochondria are able to discharge a range of intracel- lular signals including reactive oxygen species (ROS), mitochondrial DNA (mtDNA) and specific proteins, thus operating as fundamental hubs of a wide array of signalling pathways (Galluzzi et\\ua0al., 2012)
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014
restore the assembly of complex III in yeast cells mutated in the AAA domain of Bcs1. Unexpectedl... more restore the assembly of complex III in yeast cells mutated in the AAA domain of Bcs1. Unexpectedly, the first class of compensatory mutations mainly target the mitochondrial ATP synthase, leading to a strong decrease in the ATP hydrolysis activity while maintaining a sufficient level of ATP synthesis to sustain respiratory growth. We propose that by reducing ATP hydrolysis by the ATP synthase, the compensatory mutations increase the concentration of ATP in mitochondria, thereby increasing the ATP hydrolysis activity of the mutated Bcs1p and allowing it to recover its chaperon function.
It has widely been established that Cl- transport is defective in cystic fibrosis fibroblasts. In... more It has widely been established that Cl- transport is defective in cystic fibrosis fibroblasts. In the present study, the effect of elevation of intracellular concentration of cyclic AMP and calcium on the efflux of Cl- from human fibroblasts has been investigated. Cyclic AMP analogs (8-bromo cAMP and dibutyryl cAMP) and a beta agonist (isoproterenol) induced only a weak stimulation (5-10%) of Cl- efflux. Conversely, elevation of cytoplasmic calcium concentration produced by addition of the Ca2+ ionophore A23187 in the efflux medium, did not affect Cl- efflux. Our data indicate that the response of Cl- efflux to elevation of cAMP and calcium is similar in normal and cystic fibrosis fibroblasts. Exposure to hypotonic medium induced a significant stimulation of Cl- efflux in fibroblasts from both normal and cystic fibrosis individuals. Substitution of Cl- in the medium by gluconate and the subsequent addition of furosemide did not inhibit the effect of hypotonicity, indicating the involvement of a conductive pathway for Cl- transport, which was insensitive to oligomicin C.
Comparative Biochemistry and Physiology Part A: Physiology, 1988
This study validates a novel approach to predict speech intelligibility for Cochlear Implant user... more This study validates a novel approach to predict speech intelligibility for Cochlear Implant users (CIs) in reverberant environments. More specifically, we explore the use of existing objective quality and intelligibility metrics, applied directly to vocoded speech degraded by room reverberation, here assessed at ten different reverberation time (RT60) values: 0 s, 0.4 s-1.0 s (0.1 s increments), 1.5 s and 2 s. Eight objective speech intelligibility predictors (SIPs) were investigated in this study. Of these, two were non-intrusive (i.e. did not require a reference signal) audio quality measures, four were intrusive, and two were intrusive speech intelligibility indexes. Three types of vocoders were implemented to examine how speech intelligibility predictions depended on the vocoder type. These were: noise-excited vocoder, tone-excited vocoder and a FFT-based N-of-M vocoder. Experimental results show that several intrusive quality and intelligibility measures were highly correlated with exponentially fit CI intelligibility data. On the other hand, only a recently-developed non-intrusive measure showed high correlations. These evaluations suggest that CI intelligibility may be accurately assessed via objective metrics applied to vocoded speech, thus may reduce the need for expensive and timeconsuming listening tests.
Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, i... more Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, is associated with mitochondrial DNA pathogenic point mutations affecting different subunits of complex I. We here report that osteosarcoma-derived cytoplasmic hybrids (cybrid) cell lines harboring one of the three most frequent LHON pathogenic mutations, at positions 11778/ND4, 3460/ND1, and 14484/ND6, undergo cell death when galactose replaces glucose in the medium, contrary to control cybrids that maintain some growth capabilities. This is a well known way to produce a metabolic stress, forcing the cells to rely on the mitochondrial respiratory chain to produce ATP. We demonstrate that LHON cybrid cell death is apoptotic, showing chromatin condensation and nuclear DNA laddering. Moreover, we also document the mitochondrial involvement in the activation of the apoptotic cascade, as shown by the increased release of cytochrome c into the cytosol in LHON cybrid cells as compared with controls. Cybrids bearing the 3460/ND1 and 14484/ND6 mutations seemed more readily prone to undergo apoptosis as compared with the 11778/ ND4 mutation. In conclusion, LHON cybrid cells forced by the reduced rate of glycolytic flux to utilize oxidative metabolism are sensitized to an apoptotic death through a mechanism involving mitochondria.
A disruptive frameshift mtDNA mutation affecting the ND5 subunit of complex I is present in homop... more A disruptive frameshift mtDNA mutation affecting the ND5 subunit of complex I is present in homoplasmy in a nasopharyngeal oncocytic tumor and inherited as a heteroplasmic germline mutation recurring in two of the patient's siblings. Homoplasmic ND5 mutation in the tumor correlates with lack of the ND6 subunit, suggesting complex I disassembly. A few oncocytic areas, expressing ND6 and heteroplasmic for the ND5 mutation, harbor a de novo homoplasmic ND1 mutation. Since shift to homoplasmy of ND1 and ND5 mutations occurs exclusively in tumor cells, we conclude that complex I mutations may have a selective advantage and accompany oncocytic transformation.
Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the funct... more Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe exercise intolerance and multisystem manifestations. In this study, we characterized the biochemical properties of cybrids carrying this mutation and report that the homoplasmic p.278Y>C mutation caused a dramatic reduction in the CIII activity and in CIII-driven mitochondrial ATP synthesis. However, the CI, CI 1 CIII and CII 1 CIII activities and the rate of ATP synthesis driven by the CI or CII substrate were only partially reduced or unaffected. Consistent with these findings, mutated cybrids maintained the mitochondrial membrane potential in the presence of oligomycin, indicating that it originated from the respiratory electron transport chain. The p.278Y>C mutation enhanced superoxide production, as indicated by direct measurements in mitochondria and by the imbalance of glutathione homeostasis in intact cybrids. Remarkably, although the assembly of CI or CIII was not affected, the examination of respiratory supercomplexes revealed that the amounts of CIII dimer and III 2 IV 1 were reduced, whereas those of I 1 III 2 IV n slightly increased. We therefore suggest that the deleterious effects of p.278Y>C mutation on cytochrome b are palliated when CIII is assembled into the supercomplexes I 1 III 2 IV n , in contrast to when it is found alone. These findings underline the importance of supramolecular interactions between complexes for maintaining a basal respiratory chain activity and shed light to the molecular basis of disease manifestations associated with this mutation.
Previous studies have suggested an increased chloride membrane permeability in Duchenne muscular ... more Previous studies have suggested an increased chloride membrane permeability in Duchenne muscular dystrophy (DMD) fibroblasts. We report that an increased chloride efflux with respect to controls is present not only in fibroblasts from DMD, but also from two other X-linked muscular dystrophies, Becker and Emery-Dreifuss, as well as in clones from DMD carrier females. The latter observation suggests that, at least in DMD, the increased chloride efflux phenotype might be subject to iyonization.
Oncocytic tumors are characterized by cells with an aberrant accumulation of mitochondria. To ass... more 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.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2002
The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma ce... more The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2P,7P-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl 3 and Na gradients, both in HEPES-and HCO 3 3 /CO 2-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4P-diisothiocyanate dihydrostilbene-2,2P-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na /H exchanger. In addition to the amiloride-sensitive Na /H exchanger, another H extruding mechanism was detected in U-2 OS cells, the Na-dependent HCO 3 3 /Cl 3 exchanger. No significant difference in resting pH i and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V max and KP values for intracellular [H ] of the Na /H exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH i regulation in U-2 OS cells, the kinetic parameters of the Na /H exchanger were altered, suggesting a functional relationship between the two membrane proteins.
In hypotonic media, most mammalian cells initially swell as nearly perfect osmometers, but subseq... more In hypotonic media, most mammalian cells initially swell as nearly perfect osmometers, but subsequently regulate their volume (regulatory volume decrease, RVD). The shrinkage is produced by net loss of KCI combined with the exit of osmotic water. The general prevalence of this phenomenon suggests that it has physiologic importance; however, the underlying molecular mechanisms may be different in different cell types. In fact, in most of the red cells, the RVD seems to involve activation of a KC1 cotransport,I4 except for Amphiuma red cells in
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
Respiratory complex III (CIII) is the central complex of the respiratory chain. In its native for... more Respiratory complex III (CIII) is the central complex of the respiratory chain. In its native form, CIII is dimeric, and is closely associated in varying proportions with CI and CIV to form supramolecular structures, called supercomplexes [1]. The occurrence of such supercomplexes as structural and functional entities has been recently documented [1,2]. However, the physiological implications of such specific supramolecular interactions are not yet fully understood. Mutations in cytochrome b, which is the only CIII subunit encoded by the mitochondrial genome, are among the least common abnormalities identified to date in humans [3]. Recent studies on the bacterial enzyme proposed that specific amino acids at the Q o site of CIII might provide protective mechanisms against oxidative damage. Interestingly, the conserved Tyr residue at position 302 of Rhodobacter capsulatus cytochrome b is critical for this process [4]. The same mutation at position 278 of human cytochrome b (p.Y278C, m.15579ANG) has also been encountered in a patient with severe exercise intolerance and multisystem disorder [5]. Here we have utilized cybrids bearing the homoplasmic p.Y278C mutation to dissect the biochemical alterations in CIII activity, ROS production and supercomplexes assembly/stability. Despite a dramatic reduction in CIII activity, and in CIII-driven ATP synthesis, the CI and I + III activities were less affected, and the rate of ATP synthesis driven by CI or CII substrates was only partially reduced. Accordingly, mutated cybrids maintained the mitochondrial potential in the presence of oligomycin. Remarkably, the p.Y278C mutation enhanced superoxide production and perturbed glutathione homeostasis. Finally, examination of respiratory supercomplexes revealed that dimeric CIII and III 2 IV 1 were markedly decreased, whereas supercomplexes I 1 III 2 IV n increased. These findings suggest that the deleterious effects of cytochrome b p.Y278C mutation are mitigated when CIII is assembled into the supercomplexes I 1 III 2 IV n and underline the importance of supramolecular interactions between respiratory complexes on disease manifestations associated with this mutation.
OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plu... more OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plus, pathologies for which there is no established cure. We used a ‘drug repurposing’ approach to identify FDA-approved molecules able to rescue the mitochondrial dysfunctions induced by OPA1 mutations. We screened two different chemical libraries by using two yeast strains carrying the mgm1I322M and the chim3P646L mutations, identifying 26 drugs able to rescue their oxidative growth phenotype. Six of them, able to reduce the mitochondrial DNA instability in yeast, have been then tested in Opa1 deleted mouse embryonic fibroblasts expressing the human OPA1 isoform 1 bearing the R445H and D603H mutations. Some of these molecules were able to ameliorate the energetic functions and/or the mitochondrial network morphology, depending on the type of OPA1 mutation. The final validation has been performed in patients’ fibroblasts, allowing to select the most effective molecules. Our current results...
Redox Proteins in Supercomplexes and Signalosomes, 2015
In eukaryotic cells, mitochondria play the fundamental role of ATP production during the process ... more In eukaryotic cells, mitochondria play the fundamental role of ATP production during the process of oxidative phosphorylation (OXPHOS). However, these cytosolic organelles also have several other important physiological functions, including sugar and fatty acid catabo- lism, amino acid metabolism, buffering of the cytosolic calcium concentration (Rizzuto et\\ua0al., 2012), regulation and execution of different types of cell death (Galluzzi et\\ua0al., 2012) and arrangement of adaptive responses to perturbations of intracellular homeostasis (Liesa and Shirihai, 2013). Furthermore, mitochondria are able to discharge a range of intracel- lular signals including reactive oxygen species (ROS), mitochondrial DNA (mtDNA) and specific proteins, thus operating as fundamental hubs of a wide array of signalling pathways (Galluzzi et\\ua0al., 2012)
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014
restore the assembly of complex III in yeast cells mutated in the AAA domain of Bcs1. Unexpectedl... more restore the assembly of complex III in yeast cells mutated in the AAA domain of Bcs1. Unexpectedly, the first class of compensatory mutations mainly target the mitochondrial ATP synthase, leading to a strong decrease in the ATP hydrolysis activity while maintaining a sufficient level of ATP synthesis to sustain respiratory growth. We propose that by reducing ATP hydrolysis by the ATP synthase, the compensatory mutations increase the concentration of ATP in mitochondria, thereby increasing the ATP hydrolysis activity of the mutated Bcs1p and allowing it to recover its chaperon function.
It has widely been established that Cl- transport is defective in cystic fibrosis fibroblasts. In... more It has widely been established that Cl- transport is defective in cystic fibrosis fibroblasts. In the present study, the effect of elevation of intracellular concentration of cyclic AMP and calcium on the efflux of Cl- from human fibroblasts has been investigated. Cyclic AMP analogs (8-bromo cAMP and dibutyryl cAMP) and a beta agonist (isoproterenol) induced only a weak stimulation (5-10%) of Cl- efflux. Conversely, elevation of cytoplasmic calcium concentration produced by addition of the Ca2+ ionophore A23187 in the efflux medium, did not affect Cl- efflux. Our data indicate that the response of Cl- efflux to elevation of cAMP and calcium is similar in normal and cystic fibrosis fibroblasts. Exposure to hypotonic medium induced a significant stimulation of Cl- efflux in fibroblasts from both normal and cystic fibrosis individuals. Substitution of Cl- in the medium by gluconate and the subsequent addition of furosemide did not inhibit the effect of hypotonicity, indicating the involvement of a conductive pathway for Cl- transport, which was insensitive to oligomicin C.
Comparative Biochemistry and Physiology Part A: Physiology, 1988
This study validates a novel approach to predict speech intelligibility for Cochlear Implant user... more This study validates a novel approach to predict speech intelligibility for Cochlear Implant users (CIs) in reverberant environments. More specifically, we explore the use of existing objective quality and intelligibility metrics, applied directly to vocoded speech degraded by room reverberation, here assessed at ten different reverberation time (RT60) values: 0 s, 0.4 s-1.0 s (0.1 s increments), 1.5 s and 2 s. Eight objective speech intelligibility predictors (SIPs) were investigated in this study. Of these, two were non-intrusive (i.e. did not require a reference signal) audio quality measures, four were intrusive, and two were intrusive speech intelligibility indexes. Three types of vocoders were implemented to examine how speech intelligibility predictions depended on the vocoder type. These were: noise-excited vocoder, tone-excited vocoder and a FFT-based N-of-M vocoder. Experimental results show that several intrusive quality and intelligibility measures were highly correlated with exponentially fit CI intelligibility data. On the other hand, only a recently-developed non-intrusive measure showed high correlations. These evaluations suggest that CI intelligibility may be accurately assessed via objective metrics applied to vocoded speech, thus may reduce the need for expensive and timeconsuming listening tests.
Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, i... more Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, is associated with mitochondrial DNA pathogenic point mutations affecting different subunits of complex I. We here report that osteosarcoma-derived cytoplasmic hybrids (cybrid) cell lines harboring one of the three most frequent LHON pathogenic mutations, at positions 11778/ND4, 3460/ND1, and 14484/ND6, undergo cell death when galactose replaces glucose in the medium, contrary to control cybrids that maintain some growth capabilities. This is a well known way to produce a metabolic stress, forcing the cells to rely on the mitochondrial respiratory chain to produce ATP. We demonstrate that LHON cybrid cell death is apoptotic, showing chromatin condensation and nuclear DNA laddering. Moreover, we also document the mitochondrial involvement in the activation of the apoptotic cascade, as shown by the increased release of cytochrome c into the cytosol in LHON cybrid cells as compared with controls. Cybrids bearing the 3460/ND1 and 14484/ND6 mutations seemed more readily prone to undergo apoptosis as compared with the 11778/ ND4 mutation. In conclusion, LHON cybrid cells forced by the reduced rate of glycolytic flux to utilize oxidative metabolism are sensitized to an apoptotic death through a mechanism involving mitochondria.
A disruptive frameshift mtDNA mutation affecting the ND5 subunit of complex I is present in homop... more A disruptive frameshift mtDNA mutation affecting the ND5 subunit of complex I is present in homoplasmy in a nasopharyngeal oncocytic tumor and inherited as a heteroplasmic germline mutation recurring in two of the patient's siblings. Homoplasmic ND5 mutation in the tumor correlates with lack of the ND6 subunit, suggesting complex I disassembly. A few oncocytic areas, expressing ND6 and heteroplasmic for the ND5 mutation, harbor a de novo homoplasmic ND1 mutation. Since shift to homoplasmy of ND1 and ND5 mutations occurs exclusively in tumor cells, we conclude that complex I mutations may have a selective advantage and accompany oncocytic transformation.
Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the funct... more Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe exercise intolerance and multisystem manifestations. In this study, we characterized the biochemical properties of cybrids carrying this mutation and report that the homoplasmic p.278Y>C mutation caused a dramatic reduction in the CIII activity and in CIII-driven mitochondrial ATP synthesis. However, the CI, CI 1 CIII and CII 1 CIII activities and the rate of ATP synthesis driven by the CI or CII substrate were only partially reduced or unaffected. Consistent with these findings, mutated cybrids maintained the mitochondrial membrane potential in the presence of oligomycin, indicating that it originated from the respiratory electron transport chain. The p.278Y>C mutation enhanced superoxide production, as indicated by direct measurements in mitochondria and by the imbalance of glutathione homeostasis in intact cybrids. Remarkably, although the assembly of CI or CIII was not affected, the examination of respiratory supercomplexes revealed that the amounts of CIII dimer and III 2 IV 1 were reduced, whereas those of I 1 III 2 IV n slightly increased. We therefore suggest that the deleterious effects of p.278Y>C mutation on cytochrome b are palliated when CIII is assembled into the supercomplexes I 1 III 2 IV n , in contrast to when it is found alone. These findings underline the importance of supramolecular interactions between complexes for maintaining a basal respiratory chain activity and shed light to the molecular basis of disease manifestations associated with this mutation.
Previous studies have suggested an increased chloride membrane permeability in Duchenne muscular ... more Previous studies have suggested an increased chloride membrane permeability in Duchenne muscular dystrophy (DMD) fibroblasts. We report that an increased chloride efflux with respect to controls is present not only in fibroblasts from DMD, but also from two other X-linked muscular dystrophies, Becker and Emery-Dreifuss, as well as in clones from DMD carrier females. The latter observation suggests that, at least in DMD, the increased chloride efflux phenotype might be subject to iyonization.
Oncocytic tumors are characterized by cells with an aberrant accumulation of mitochondria. To ass... more 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.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2002
The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma ce... more The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2P,7P-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl 3 and Na gradients, both in HEPES-and HCO 3 3 /CO 2-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4P-diisothiocyanate dihydrostilbene-2,2P-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na /H exchanger. In addition to the amiloride-sensitive Na /H exchanger, another H extruding mechanism was detected in U-2 OS cells, the Na-dependent HCO 3 3 /Cl 3 exchanger. No significant difference in resting pH i and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V max and KP values for intracellular [H ] of the Na /H exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH i regulation in U-2 OS cells, the kinetic parameters of the Na /H exchanger were altered, suggesting a functional relationship between the two membrane proteins.
In hypotonic media, most mammalian cells initially swell as nearly perfect osmometers, but subseq... more In hypotonic media, most mammalian cells initially swell as nearly perfect osmometers, but subsequently regulate their volume (regulatory volume decrease, RVD). The shrinkage is produced by net loss of KCI combined with the exit of osmotic water. The general prevalence of this phenomenon suggests that it has physiologic importance; however, the underlying molecular mechanisms may be different in different cell types. In fact, in most of the red cells, the RVD seems to involve activation of a KC1 cotransport,I4 except for Amphiuma red cells in
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