Publications by authors named "Cristina Ugalde"

41 Publications

SILAC-based complexome profiling dissects the structural organization of the human respiratory supercomplexes in SCAFI cells.

Biochim Biophys Acta Bioenerg 2021 Mar 13;1862(7):148414. Epub 2021 Mar 13.

Instituto de Investigación, Hospital Universitario, 12 de Octubre, Madrid 28041, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723 Madrid, Spain. Electronic address:

The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the individual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of individual MRC complexes III and IV in the minor SC III + IV, although its relevance for respirasome formation and function remains controversial. In this work, we have used SILAC-based complexome profiling to dissect the structural organization of the human MRC in HEK293T cells depleted of SCAFI (SCAFI) by CRISPR-Cas9 genome editing. SCAFI ablation led to a preferential loss of SC III + IV and of a minor subset of respirasomes without affecting OXPHOS function. Our data suggest that the loss of SCAFI-dependent respirasomes in SCAFI cells is mainly due to alterations on early stages of CI assembly, without impacting the biogenesis of complexes III and IV. Contrary to the idea of SCAFI being the main player in respirasome formation, SILAC-complexome profiling showed that, in wild-type cells, the majority of respirasomes (ca. 70%) contained COX7A2 and that these species were present at roughly the same levels when SCAFI was knocked-out. We thus demonstrate the co-existence of structurally distinct respirasomes defined by the preferential binding of complex IV via COX7A2, rather than SCAFI, in human cultured cells.
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http://dx.doi.org/10.1016/j.bbabio.2021.148414DOI Listing
March 2021

CEDAR, an online resource for the reporting and exploration of complexome profiling data.

Biochim Biophys Acta Bioenerg 2021 Mar 17;1862(7):148411. Epub 2021 Mar 17.

Center for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands. Electronic address:

Complexome profiling is an emerging 'omics' approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the "minimum information required for a complexome profiling experiment" (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.
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http://dx.doi.org/10.1016/j.bbabio.2021.148411DOI Listing
March 2021

Uniparental isodisomy as a cause of mitochondrial complex I respiratory chain disorder due to a novel splicing NDUFS4 mutation.

Mol Genet Metab 2020 11 16;131(3):341-348. Epub 2020 Oct 16.

Mitochondrial Diseases Laboratory, Hospital Universitario '12 de Octubre', Madrid, Spain; Center for Biomedical Network Research on Rare Diseases (CIBERER), Madrid, Spain; Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), Madrid, Spain. Electronic address:

Uniparental disomy (UPD) is an underestimated cause of autosomal recessive disorders. In this study, we aim to raise awareness about the possibility of UPD in mitochondrial disorders - where it is a hardly described event -, by functionally characterizing a novel variant in a structural subunit of complex I (CI) of the mitochondrial oxidative phosphorylation system. Using next-generation sequencing, we identified a new intronic homozygous c.350 + 5G > A variant in the NDUFS4 gene in a one-year-old girl (being alive at the age of 7) belonging to a non-consanguineous family presenting with encephalopathy, psychomotor delay, lactic acidosis and a single CI deficiency, a less severe phenotype than those previously reported in most NDUFS4 patients. One parent lacked the variant, and microsatellite genotyping showed complete paternal uniparental isodisomy of the non-imprinted chromosome 5. We demonstrated in patient's skeletal muscle and fibroblasts splicing abnormalities, low expression of NDUFS4, undetectable NDUFS4 protein, defects in cellular respiration (decreased oxygen consumption and ATP production), and impaired assembly or stability of mitochondrial supercomplexes containing CI. Our findings support that c.350 + 5G > A variant is pathogenic, and reinforce that UPD, although rare, should be considered as a possible cause of mitochondrial diseases in order to provide accurate genetic counselling.
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http://dx.doi.org/10.1016/j.ymgme.2020.10.008DOI Listing
November 2020

Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis.

STAR Protoc 2020 Sep 3;1(2). Epub 2020 Sep 3.

Department of Neurology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

By using negatively charged Coomassie brilliant blue G-250 dye to induce a charge shift on proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE) allows resolution of enzymatically active multiprotein complexes extracted from cellular or subcellular lysates while retaining their native conformation. BN-PAGE was first developed to analyze the size, composition, and relative abundance of the complexes and supercomplexes that form the mitochondrial respiratory chain and OXPHOS system. Here, we present a detailed protocol of BN-PAGE to obtain robust and reproducible results. For complete details on the use and execution of this protocol, please refer to Lobo-Jarne et al. (2018) and Timón-Gómez et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521667PMC
September 2020

Altered Expression Ratio of Actin-Binding Gelsolin Isoforms Is a Novel Hallmark of Mitochondrial OXPHOS Dysfunction.

Cells 2020 08 19;9(9). Epub 2020 Aug 19.

Laboratorio de Enfermedades Mitocondriales y Neurometabólicas, Instituto de Investigación Hospital 12 de Octubre (i+12), 28041 Madrid, Spain.

Mitochondrial oxidative phosphorylation (OXPHOS) defects are the primary cause of inborn errors of energy metabolism. Despite considerable progress on their genetic basis, their global pathophysiological consequences remain undefined. Previous studies reported that OXPHOS dysfunction associated with complex III deficiency exacerbated the expression and mitochondrial location of cytoskeletal gelsolin (GSN) to promote cell survival responses. In humans, besides the cytosolic isoform, GSN presents a plasma isoform secreted to extracellular environments. We analyzed the interplay between both GSN isoforms in human cellular and clinical models of OXPHOS dysfunction. Regardless of its pathogenic origin, OXPHOS dysfunction induced the physiological upregulation of cytosolic GSN in the mitochondria (mGSN), in parallel with a significant downregulation of plasma GSN (pGSN) levels. Consequently, significantly high mGSN-to-pGSN ratios were associated with OXPHOS deficiency both in human cells and blood. In contrast, control cells subjected to hydrogen peroxide or staurosporine treatments showed no correlation between oxidative stress or cell death induction and the altered levels and subcellular location of GSN isoforms, suggesting their specificity for OXPHOS dysfunction. In conclusion, a high mitochondrial-to-plasma GSN ratio represents a useful cellular indicator of OXPHOS defects, with potential use for future research of a wide range of clinical conditions with mitochondrial involvement.
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http://dx.doi.org/10.3390/cells9091922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563380PMC
August 2020

Novel Mutations Associated with OXPHOS Deficiency and Leigh Syndrome: A Second Family Report.

Genes (Basel) 2020 07 26;11(8). Epub 2020 Jul 26.

Laboratorio de Enfermedades Mitocondriales y Neurometabólicas, Instituto de Investigación Hospital 12 de Octubre (imas12), 28041 Madrid, Spain.

Leigh syndrome (LS) usually presents as an early onset mitochondrial encephalopathy characterized by bilateral symmetric lesions in the basal ganglia and cerebral stem. More than 75 genes have been associated with this condition, including genes involved in the biogenesis of mitochondrial complex I (CI). In this study, we used a next-generation sequencing (NGS) panel to identify two novel biallelic variants in the NADH:ubiquinone oxidoreductase subunit A13 () gene in a patient with isolated CI deficiency in skeletal muscle. Our patient, who represents the second family report with mutations in the CI NDUFA13 subunit, presented with LS lesions in brain magnetic resonance imaging, mild hypertrophic cardiomyopathy, and progressive spastic tetraparesis. This phenotype manifestation is different from that previously described in the first family, which was predominantly characterized by neurosensorial symptoms. Both in silico pathogenicity predictions and oxidative phosphorylation (OXPHOS) functional findings in patient's skin fibroblasts (delayed cell growth, isolated CI enzyme defect, decreased basal and maximal oxygen consumption and as well as ATP production, together with markedly diminished levels of the NDUFA13 protein, CI, and respirasomes) suggest that these novel variants in the gene are the underlying cause of the CI defect, expanding the genetic heterogeneity of LS.
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http://dx.doi.org/10.3390/genes11080855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465247PMC
July 2020

Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes.

EMBO J 2020 07 8;39(14):e103912. Epub 2020 Jun 8.

Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid, Spain.

Mitochondrial respiratory chain complexes I, III, and IV can associate into larger structures termed supercomplexes or respirasomes, thereby generating structural interdependences among the individual complexes yet to be understood. In patients, nonsense mutations in complex IV subunit genes cause severe encephalomyopathies randomly associated with pleiotropic complex I defects. Using complexome profiling and biochemical analyses, we have explored the structural rearrangements of the respiratory chain in human cell lines depleted of the catalytic complex IV subunit COX1 or COX2. In the absence of a functional complex IV holoenzyme, several supercomplex I+III species coexist, which differ in their content of COX subunits and COX7A2L/HIGD2A assembly factors. The incorporation of an atypical COX1-HIGD2A submodule attenuates supercomplex I+III turnover rate, indicating an unexpected molecular adaptation for supercomplexes stabilization that relies on the presence of COX1 independently of holo-complex IV formation. Our data set the basis for complex I structural dependence on complex IV, revealing the co-existence of alternative pathways for the biogenesis of "supercomplex-associated" versus individual complex IV, which could determine physiological adaptations under different stress and disease scenarios.
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http://dx.doi.org/10.15252/embj.2019103912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360963PMC
July 2020

Distinct Roles of Mitochondrial HIGD1A and HIGD2A in Respiratory Complex and Supercomplex Biogenesis.

Cell Rep 2020 05;31(5):107607

Department of Neurology, University of Miami Miller School of Medicine, Miami, FL 33136, USA. Electronic address:

The mitochondrial respiratory chain enzymes are organized as individual complexes and supercomplexes, whose biogenesis remains to be fully understood. To disclose the role of the human Hypoxia Inducible Gene Domain family proteins HIGD1A and HIGD2A in these processes, we generate and characterize HIGD-knockout (KO) cell lines. We show that HIGD2A controls and coordinates the modular assembly of isolated and supercomplexed complex IV (CIV) by acting on the COX3 assembly module. In contrast, HIGD1A regulates CIII and CIII-containing supercomplex biogenesis by supporting the incorporation of UQCRFS1. HIGD1A also clusters with COX4-1 and COX5A CIV subunits and, when overexpressed, suppresses the CIV biogenesis defect of HIGD2A-KO cells. We conclude that HIGD1A and HIGD2A have both independent and overlapping functions in the biogenesis of respiratory complexes and supercomplexes. Our data illuminate the existence of multiple pathways to assemble these structures by dynamic HIGD-mediated CIV biogenesis, potentially to adapt to changing environmental and nutritional conditions.
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http://dx.doi.org/10.1016/j.celrep.2020.107607DOI Listing
May 2020

Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV.

EMBO J 2020 02 8;39(3):e102817. Epub 2020 Jan 8.

Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK.

Mitochondrial respiratory chain (MRC) enzymes associate in supercomplexes (SCs) that are structurally interdependent. This may explain why defects in a single component often produce combined enzyme deficiencies in patients. A case in point is the alleged destabilization of complex I in the absence of complex III. To clarify the structural and functional relationships between complexes, we have used comprehensive proteomic, functional, and biogenetical approaches to analyze a MT-CYB-deficient human cell line. We show that the absence of complex III blocks complex I biogenesis by preventing the incorporation of the NADH module rather than decreasing its stability. In addition, complex IV subunits appeared sequestered within complex III subassemblies, leading to defective complex IV assembly as well. Therefore, we propose that complex III is central for MRC maturation and SC formation. Our results challenge the notion that SC biogenesis requires the pre-formation of fully assembled individual complexes. In contrast, they support a cooperative-assembly model in which the main role of complex III in SCs is to provide a structural and functional platform for the completion of overall MRC biogenesis.
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http://dx.doi.org/10.15252/embj.2019102817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996572PMC
February 2020

Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities.

Mol Genet Metab 2019 12 6;128(4):452-462. Epub 2019 Nov 6.

Laboratorio de Enfermedades Raras, Mitocondriales y Neuromusculares, Instituto de Investigación Hospital 12 de Octubre (i+12), 28041 Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, U723, 28029 Madrid, Spain.

Lethal neonatal encephalopathies are heterogeneous congenital disorders that can be caused by mitochondrial dysfunction. Biallelic large deletions in the contiguous ATAD3B and ATAD3A genes, encoding mitochondrial inner membrane ATPases of unknown function, as well as compound heterozygous nonsense and missense mutations in the ATAD3A gene have been recently associated with fatal neonatal cerebellar hypoplasia. In this work, whole exome sequencing (WES) identified the novel homozygous variant c.1217 T > G in ATAD3A, predicting a p.(Leu406Arg) substitution, in four siblings from a consanguineous family presenting with fatal neonatal cerebellar hypoplasia, seizures, axial hypotonia, hypertrophic cardiomyopathy, hepatomegaly, congenital cataract, and dysmorphic facies. Biochemical phenotypes of the patients included hyperlactatemia and hypocholesterolemia. Healthy siblings and parents were heterozygous for this variant, which is predicted to introduce a polar chain within the catalytic domain of ATAD3A that shortens its beta-sheet structure, presumably affecting protein stability. Accordingly, patient's fibroblasts with the homozygous variant displayed a specific reduction in ATAD3A protein levels associated with profound ultrastructural alterations of mitochondrial cristae and morphology. Our findings exclude the causative role of ATAD3B on this severe phenotype, expand the phenotypical spectrum of ATAD3A pathogenic variants and emphasize the vital role of ATAD3A in mitochondrial biogenesis.
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http://dx.doi.org/10.1016/j.ymgme.2019.10.012DOI Listing
December 2019

Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics.

Cell Rep 2018 11;25(7):1786-1799.e4

Department of Neurology, University of Miami Miller School of Medicine, Miami, FL 33136, USA; Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA. Electronic address:

The mitochondrial respiratory chain is organized in a dynamic set of supercomplexes (SCs). The COX7A2L protein is essential for mammalian SC III+IV assembly. However, its function in respirasome (SCs I+III+IV) biogenesis remains controversial. To unambiguously determine the COX7A2L role, we generated COX7A2L-knockout (COX7A2L-KO) HEK293T and U87 cells. COX7A2L-KO cells lack SC III+IV but have enhanced complex III steady-state levels, activity, and assembly rate, normal de novo complex IV biogenesis, and delayed respirasome formation. Nonetheless, the KOs have normal respirasome steady-state levels, and only larger structures (SCs I+III+IV or megacomplexes) were undetected. Functional substrate-driven competition assays showed normal mitochondrial respiration in COX7A2L-KO cells in standard and nutritional-, environmental-, and oxidative-stress-challenging conditions. We conclude that COX7A2L establishes a regulatory checkpoint for the biogenesis of CIII and specific SCs, but the COX7A2L-dependent MRC remodeling is essential neither to maintain mitochondrial bioenergetics nor to cope with acute cellular stresses.
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http://dx.doi.org/10.1016/j.celrep.2018.10.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286155PMC
November 2018

Respiratory chain supercomplexes: Structures, function and biogenesis.

Semin Cell Dev Biol 2018 04 23;76:179-190. Epub 2017 Jul 23.

Instituto de Investigación Hospital 12 de Octubre (i+12), Madrid 28041, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Madrid 28029, Spain. Electronic address:

Over the past sixty years, researchers have made outmost efforts to clarify the structural organization and functional regulation of the complexes that configure the mitochondrial respiratory chain. As a result, the entire composition of each individual complex is practically known and, aided by notable structural advances in mammals, it is now widely accepted that these complexes stablish interactions to form higher-order supramolecular structures called supercomplexes and respirasomes. The mechanistic models and players that regulate the function and biogenesis of such superstructures are still under intense debate, and represent one of the hottest topics of the mitochondrial research field at present. Noteworthy, understanding the pathways involved in the assembly and organization of respiratory chain complexes and supercomplexes is of high biomedical relevance because molecular alterations in these pathways frequently result in severe mitochondrial disorders. The purpose of this review is to update the structural, biogenetic and functional knowledge about the respiratory chain supercomplexes and assembly factors involved in their formation, with special emphasis on their implications in mitochondrial disease. Thanks to the integrated data resulting from recent structural, biochemical and genetic approaches in diverse biological systems, the regulation of the respiratory chain function arises at multiple levels of complexity.
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http://dx.doi.org/10.1016/j.semcdb.2017.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780262PMC
April 2018

Respiratory chain enzyme deficiency induces mitochondrial location of actin-binding gelsolin to modulate the oligomerization of VDAC complexes and cell survival.

Hum Mol Genet 2017 07;26(13):2493-2506

Instituto de Investigación, Hospital Universitario 12 de Octubre, Madrid 28041, Spain.

Despite considerable knowledge on the genetic basis of mitochondrial disorders, their pathophysiological consequences remain poorly understood. We previously used two-dimensional difference gel electrophoresis analyses to define a protein profile characteristic for respiratory chain complex III-deficiency that included a significant overexpression of cytosolic gelsolin (GSN), a cytoskeletal protein that regulates the severing and capping of the actin filaments. Biochemical and immunofluorescence assays confirmed a specific increase of GSN levels in the mitochondria from patients' fibroblasts and from transmitochondrial cybrids with complex III assembly defects. A similar effect was obtained in control cells upon treatment with antimycin A in a dose-dependent manner, showing that the enzymatic inhibition of complex III is sufficient to promote the mitochondrial localization of GSN. Mitochondrial subfractionation showed the localization of GSN to the mitochondrial outer membrane, where it interacts with the voltage-dependent anion channel protein 1 (VDAC1). In control cells, VDAC1 was present in five stable oligomeric complexes, which showed increased levels and a modified distribution pattern in the complex III-deficient cybrids. Downregulation of GSN expression induced cell death in both cell types, in parallel with the specific accumulation of VDAC1 dimers and the release of mitochondrial cytochrome c into the cytosol, indicating a role for GSN in the oligomerization of VDAC complexes and in the prevention of apoptosis. Our results demonstrate that respiratory chain complex III dysfunction induces the physiological upregulation and mitochondrial location of GSN, probably to promote cell survival responses through the modulation of the oligomeric state of the VDAC complexes.
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http://dx.doi.org/10.1093/hmg/ddx144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192415PMC
July 2017

COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation.

Cell Rep 2016 08 18;16(9):2387-98. Epub 2016 Aug 18.

Instituto de Investigación, Hospital Universitario 12 de Octubre (i+12), Madrid 28041, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Madrid 28029, Spain. Electronic address:

Mitochondrial respiratory chain (MRC) complexes I, III, and IV associate into a variety of supramolecular structures known as supercomplexes and respirasomes. While COX7A2L was originally described as a supercomplex-specific factor responsible for the dynamic association of complex IV into these structures to adapt MRC function to metabolic variations, this role has been disputed. Here, we further examine the functional significance of COX7A2L in the structural organization of the mammalian respiratory chain. As in the mouse, human COX7A2L binds primarily to free mitochondrial complex III and, to a minor extent, to complex IV to specifically promote the stabilization of the III2+IV supercomplex without affecting respirasome formation. Furthermore, COX7A2L does not affect the biogenesis, stabilization, and function of the individual oxidative phosphorylation complexes. These data show that independent regulatory mechanisms for the biogenesis and turnover of different MRC supercomplex structures co-exist.
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http://dx.doi.org/10.1016/j.celrep.2016.07.081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007171PMC
August 2016

Differential proteomic profiling unveils new molecular mechanisms associated with mitochondrial complex III deficiency.

J Proteomics 2015 Jan 18;113:38-56. Epub 2014 Sep 18.

Instituto de Investigación, Hospital Universitario 12 de Octubre, Madrid 28041, Spain.

Unlabelled: We have analyzed the cellular pathways and metabolic adaptations that take place in primary skin fibroblasts from patients with mutations in BCS1L, a major genetic cause of mitochondrial complex III enzyme deficiency. Mutant fibroblasts exhibited low oxygen consumption rates and intracellular ATP levels, indicating that the main altered molecular event probably is a limited respiration-coupled ATP production through the OXPHOS system. Two-dimensional DIGE and MALDI-TOF/TOF mass spectrometry analyses unambiguously identified 39 proteins whose expression was significantly altered in complex III-deficient fibroblasts. Extensive statistical and cluster analyses revealed a protein profile characteristic for the BCS1L mutant fibroblasts that included alterations in energy metabolism, cell signaling and gene expression regulation, cytoskeleton formation and maintenance, and intracellular stress responses. The physiological validation of the predicted functional adaptations of human cultured fibroblasts to complex III deficiency confirmed the up-regulation of glycolytic enzyme activities and the accumulation of branched-chain among other amino acids, suggesting the activation of anaerobic glycolysis and cellular catabolic states, in particular protein catabolism, together with autophagy as adaptive responses to mitochondrial respiratory chain dysfunction and ATP deficiency. Our data point to an overall metabolic and genetic reprogramming that could contribute to explain the clinical manifestations of complex III deficiency in patients.

Biological Significance: Despite considerable knowledge about their genetic origins, the pathophysiological mechanisms that contribute to the clinical manifestations of mitochondrial disorders remain poorly understood. We have investigated the molecular pathways and metabolic adaptations that take place in primary skin fibroblasts from patients with mutations in the BCS1L gene, a primary cause of mitochondrial complex III enzyme deficiency. Two-dimensional DIGE together with MALDI-TOF/TOF mass spectrometry and physiological validation analyses revealed a significant metabolic and genetic reprogramming as an adaptive response to mitochondrial respiratory chain dysfunction. Our data provide information about specific protein targets that regulate the transmitochondrial functional responses to complex III deficiency, thereby opening new doors for future research.
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http://dx.doi.org/10.1016/j.jprot.2014.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259860PMC
January 2015

Bulk autophagy, but not mitophagy, is increased in cellular model of mitochondrial disease.

Biochim Biophys Acta 2014 Jul 2;1842(7):1059-70. Epub 2014 Apr 2.

Mitochondrial and Neuromuscular Diseases Laboratory, Hospital Universitario 12 de Octubre Research Institute (i+12), Madrid, Spain; Spanish Network for Biomedical Research in Rare Diseases (CIBERER), U723, Spain.

Oxidative phosphorylation system (OXPHOS) deficiencies are rare diseases but constitute the most frequent inborn errors of metabolism. We analyzed the autophagy route in 11 skin fibroblast cultures derived from patients with well characterized and distinct OXPHOS defects. Mitochondrial membrane potential determination revealed a tendency to decrease in 5 patients' cells but reached statistical significance only in 2 of them. The remaining cells showed either no change or a slight increase in this parameter. Colocalization analysis of mitochondria and autophagosomes failed to show evidence of increased selective elimination of mitochondria but revealed more intense autophagosome staining in patients' fibroblasts compared with controls. Despite the absence of increased mitophagy, Parkin recruitment to mitochondria was detected in both controls' and patients' cells and was slightly higher in cells harboring complex I defects. Western blot analysis of the autophagosome marker LC3B, confirmed significantly higher levels of the protein bound to autophagosomes, LC3B-II, in patients' cells, suggesting an increased bulk autophagy in OXPHOS defective fibroblasts. Inhibition of lysosomal proteases caused significant accumulation of LC3B-II in control cells, whereas in patients' cells this phenomenon was less pronounced. Electron microscopy studies showed higher content of late autophagic vacuoles and lysosomes in OXPHOS defective cells, accompanied by higher levels of the lysosomal marker LAMP-1. Our findings suggest that in OXPHOS deficient fibroblasts autophagic flux could be partially hampered leading to an accumulation of autophagic vacuoles and lysosomes.
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http://dx.doi.org/10.1016/j.bbadis.2014.03.013DOI Listing
July 2014

I function, therefore I am: overcoming skepticism about mitochondrial supercomplexes.

Cell Metab 2013 Aug;18(2):147-9

Department of Neurology, University of Miami Miller School of Medicine, FL 33136, USA.

The mitochondrial respiratory chain is believed to dynamically arrange in suprastructures known as supercomplexes or respirasomes, though their function remains elusive. A recent study in Science (Lapuente-Brun et al., 2013) now reports that dynamic supercomplex assembly determines electron flux from different substrates through the respiratory chain.
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http://dx.doi.org/10.1016/j.cmet.2013.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3912836PMC
August 2013

Coiled coil domain-containing protein 56 (CCDC56) is a novel mitochondrial protein essential for cytochrome c oxidase function.

J Biol Chem 2012 Jul 18;287(29):24174-85. Epub 2012 May 18.

Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols" Universidad Autónoma de Madrid (UAM)-Consejo Superior de Investigaciones Científicas (CSIC), Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER) Facultad de Medicina, 28029 Madrid, Spain.

In Drosophila melanogaster, the mitochondrial transcription factor B1 (d-mtTFB1) transcript contains in its 5'-untranslated region a conserved upstream open reading frame denoted as CG42630 in FlyBase. We demonstrate that CG42630 encodes a novel protein, the coiled coil domain-containing protein 56 (CCDC56), conserved in metazoans. We show that Drosophila CCDC56 protein localizes to mitochondria and contains 87 amino acids in flies and 106 in humans with the two proteins sharing 42% amino acid identity. We show by rapid amplification of cDNA ends and Northern blotting that Drosophila CCDC56 protein and mtTFB1 are encoded on a bona fide bicistronic transcript. We report the generation and characterization of two ccdc56 knock-out lines in Drosophila carrying the ccdc56(D6) and ccdc56(D11) alleles. Lack of the CCDC56 protein in flies induces a developmental delay and 100% lethality by arrest of larval development at the third instar. ccdc56 knock-out larvae show a significant decrease in the level of fully assembled cytochrome c oxidase (COX) and in its activity, suggesting a defect in complex assembly; the activity of the other oxidative phosphorylation complexes remained either unaffected or increased in the ccdc56 knock-out larvae. The lethal phenotype and the decrease in COX were partially rescued by reintroduction of a wild-type UAS-ccdc56 transgene. These results indicate an important role for CCDC56 in the oxidative phosphorylation system and in particular in COX function required for proper development in D. melanogaster. We propose CCDC56 as a candidate factor required for COX biogenesis/assembly.
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http://dx.doi.org/10.1074/jbc.M112.343764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397844PMC
July 2012

Mitochondrial respiratory chain dysfunction: implications in neurodegeneration.

Free Radic Biol Med 2012 Aug 14;53(3):595-609. Epub 2012 May 14.

Laboratorio de Enfermedades Raras: Mitocondriales y Neuromusculares, Instituto de Investigación Hospital Universitario 12 de Octubre (i+12), Madrid, Spain.

For decades mitochondria have been considered static round-shaped organelles in charge of energy production. In contrast, they are highly dynamic cellular components that undergo continuous cycles of fusion and fission influenced, for instance, by oxidative stress, cellular energy requirements, or the cell cycle state. New important functions beyond energy production have been attributed to mitochondria, such as the regulation of cell survival, because of their role in the modulation of apoptosis, autophagy, and aging. Primary mitochondrial diseases due to mutations in genes involved in these new mitochondrial functions and the implication of mitochondrial dysfunction in multifactorial human pathologies such as cancer, Alzheimer and Parkinson diseases, or diabetes has been demonstrated. Therefore, mitochondria are set at a central point of the equilibrium between health and disease, and a better understanding of mitochondrial functions will open new fields for exploring the roles of these mitochondrial pathways in human pathologies. This review dissects the relationships between activity and assembly defects of the mitochondrial respiratory chain, oxidative damage, and alterations in mitochondrial dynamics, with special focus on their implications for neurodegeneration.
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http://dx.doi.org/10.1016/j.freeradbiomed.2012.05.009DOI Listing
August 2012

Mitochondrial complex I plays an essential role in human respirasome assembly.

Cell Metab 2012 Mar 16;15(3):324-35. Epub 2012 Feb 16.

Instituto de Investigación, Hospital Universitario 12 de Octubre, Madrid 28041, Spain.

The biogenesis and function of the mitochondrial respiratory chain (RC) involve the organization of RC enzyme complexes in supercomplexes or respirasomes through an unknown biosynthetic process. This leads to structural interdependences between RC complexes, which are highly relevant from biological and biomedical perspectives, because RC defects often lead to severe neuromuscular disorders. We show that in human cells, respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of complexes III and IV subunits, rather than originating from the association of preassembled individual holoenzymes. The process ends with the incorporation of complex I NADH dehydrogenase catalytic module, which leads to the respirasome activation. While complexes III and IV assemble either as free holoenzymes or by incorporation of free subunits into supercomplexes, the respirasomes constitute the structural units where complex I is assembled and activated, thus explaining the significance of the respirasomes for RC function.
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http://dx.doi.org/10.1016/j.cmet.2012.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318979PMC
March 2012

Impact of the mitochondrial genetic background in complex III deficiency.

PLoS One 2010 Sep 17;5(9). Epub 2010 Sep 17.

Centro de Investigación, Hospital Universitario 12 de Octubre, Madrid, Spain.

Background: In recent years clinical evidence has emphasized the importance of the mtDNA genetic background that hosts a primary pathogenic mutation in the clinical expression of mitochondrial disorders, but little experimental confirmation has been provided. We have analyzed the pathogenic role of a novel homoplasmic mutation (m.15533 A>G) in the cytochrome b (MT-CYB) gene in a patient presenting with lactic acidosis, seizures, mild mental delay, and behaviour abnormalities.

Methodology: Spectrophotometric analyses of the respiratory chain enzyme activities were performed in different tissues, the whole muscle mitochondrial DNA of the patient was sequenced, and the novel mutation was confirmed by PCR-RFLP. Transmitochondrial cybrids were constructed to confirm the pathogenicity of the mutation, and assembly/stability studies were carried out in fibroblasts and cybrids by means of mitochondrial translation inhibition in combination with blue native gel electrophoresis.

Principal Findings: Biochemical analyses revealed a decrease in respiratory chain complex III activity in patient's skeletal muscle, and a combined enzyme defect of complexes III and IV in fibroblasts. Mutant transmitochondrial cybrids restored normal enzyme activities and steady-state protein levels, the mutation was mildly conserved along evolution, and the proband's mother and maternal aunt, both clinically unaffected, also harboured the homoplasmic mutation. These data suggested a nuclear genetic origin of the disease. However, by forcing the de novo functioning of the OXPHOS system, a severe delay in the biogenesis of the respiratory chain complexes was observed in the mutants, which demonstrated a direct functional effect of the mitochondrial genetic background.

Conclusions: Our results point to possible pitfalls in the detection of pathogenic mitochondrial mutations, and highlight the role of the genetic mtDNA background in the development of mitochondrial disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012801PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2941448PMC
September 2010

Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency.

Hum Mutat 2010 Aug;31(8):930-41

Centro de Investigación, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain.

Mutations in BCS1L, an assembly factor that facilitates the insertion of the catalytic Rieske Iron-Sulfur subunit into respiratory chain complex III, result in a wide variety of clinical phenotypes that range from the relatively mild Björnstad syndrome to the severe GRACILE syndrome. To better understand the pathophysiological consequences of such mutations, we studied fibroblasts from six complex III-deficient patients harboring mutations in the BCS1L gene. Cells from patients with the most severe clinical phenotypes exhibited slow growth rates in glucose medium, variable combined enzyme deficiencies, and assembly defects of respiratory chain complexes I, III, and IV, increased H(2)O(2) levels, unbalanced expression of the cellular antioxidant defenses, and apoptotic cell death. In addition, all patients showed cytosolic accumulation of the BCS1L protein, suggestive of an impaired mitochondrial import, assembly or stability defects of the BCS1L complex, fragmentation of the mitochondrial networks, and decreased MFN2 protein levels. The observed structural alterations were independent of the respiratory chain function and ROS production. Our results provide new insights into the role of pathogenic BCS1L mutations in mitochondrial function and dynamics.
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http://dx.doi.org/10.1002/humu.21294DOI Listing
August 2010

Gelsolin restores A beta-induced alterations in choroid plexus epithelium.

J Biomed Biotechnol 2010 25;2010:805405. Epub 2010 Mar 25.

Neuroscience Laboratory, Research Center, Hospital 12 de Octubre, Avenida de Córdoba, 28041 Madrid, Spain.

Histologically, Alzheimer's disease (AD) is characterized by senile plaques and cerebrovascular amyloid deposits. In previous studies we demonstrated that in AD patients, amyloid-beta (A beta) peptide also accumulates in choroid plexus, and that this process is associated with mitochondrial dysfunction and epithelial cell death. However, the molecular mechanisms underlying A beta accumulation at the choroid plexus epithelium remain unclear. A beta clearance, from the brain to the blood, involves A beta carrier proteins that bind to megalin, including gelsolin, a protein produced specifically by the choroid plexus epithelial cells. In this study, we show that treatment with gelsolin reduces A beta-induced cytoskeletal disruption of blood-cerebrospinal fluid (CSF) barrier at the choroid plexus. Additionally, our results demonstrate that gelsolin plays an important role in decreasing A beta-induced cytotoxicity by inhibiting nitric oxide production and apoptotic mitochondrial changes. Taken together, these findings make gelsolin an appealing tool for the prophylactic treatment of AD.
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http://dx.doi.org/10.1155/2010/805405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2847388PMC
July 2010

Marked mitochondrial DNA depletion associated with a novel SUCLG1 gene mutation resulting in lethal neonatal acidosis, multi-organ failure, and interrupted aortic arch.

Mitochondrion 2010 Jun 19;10(4):362-8. Epub 2010 Mar 19.

Laboratorio de enfermedades mitocondriales, Centro de Investigación, Hospital Universitario 12 de Octubre, Madrid, Spain.

The aim of this study was to identify the causative genetic lesion in two apparently unrelated newborns having lethal lactic acidosis, multi-organ failure and congenital malformations including interrupted aortic arch, who exhibited mild methylmalonic aciduria, combined mitochondrial respiratory chain deficiency, and marked muscle mitochondrial DNA depletion. A novel mutation in the SUCLG1 gene was identified. Phenotype severity in Succinate-CoA ligase dysfunction appears to be more correlated to the muscle mtDNA content than to the tissue distribution of the heterodimer subunits. Prominent impairment of mitochondrial respiratory chain may result in deep ravages in developmental tissues leading to multiple organ failure and malformations.
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http://dx.doi.org/10.1016/j.mito.2010.03.003DOI Listing
June 2010

Cytoplasmic gelsolin increases mitochondrial activity and reduces Abeta burden in a mouse model of Alzheimer's disease.

Neurobiol Dis 2009 Oct 14;36(1):42-50. Epub 2009 Jul 14.

Neuroscience Laboratory, Research Center, Hospital 12 de Octubre, Madrid, Spain.

Accumulation of amyloid-beta (Abeta) peptides is thought to be a critical event in the pathology of Alzheimer's disease (AD), because they induce multiple neurotoxic effects, including mitochondrial dysfunction and apoptotic cell death. Therefore the reduction of Abeta is considered a primary therapeutic target. Gelsolin, an Abeta binding protein, has been shown to inhibit apoptosis, although the underlying mechanism is unclear. To clarify these effects, we manipulated cytoplasmic gelsolin levels through viral-directed overexpression in the brain of APP/Ps1 transgenic mice. We observed that gelsolin reduces brain Abeta burden in the APP/Ps1 mice, possibly by enhancing Abeta clearance via megalin. The reduction in brain Abeta levels was accompanied by an inhibition of nitric oxide production and cell death, not only in the choroid plexus but also in the cerebral cortex. Notably, overexpressed gelsolin restored the impaired mitochondrial activity in the APP/Ps1 mice, resulting in the increase of cytochrome c oxidase activity. By contrast, RNA interference to block gelsolin expression, confirmed that cytoplasmic gelsolin acts as a modulator of brain Abeta levels and its neurotoxic effects. We conclude that gelsolin might prevent brain amyloidosis and Abeta-induced apoptotic mitochondrial changes. These findings make cytoplasmic gelsolin a potential therapeutic strategy in AD.
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http://dx.doi.org/10.1016/j.nbd.2009.06.018DOI Listing
October 2009

Pathogenic mutations in the 5' untranslated region of BCS1L mRNA in mitochondrial complex III deficiency.

Mitochondrion 2009 Sep 21;9(5):299-305. Epub 2009 Apr 21.

Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Spain.

Mutations in the assembly chaperone BCS1L constitute a major cause of mitochondrial complex III deficiency. We studied the presence of BCS1L mutations in a complex III-deficient patient with metabolic acidosis, liver failure, and tubulopathy. A previously reported mutation, p.R56X, was identified in one BCS1L allele, and two novel heterozygous mutations, g.1181A>G and g.1164C>G, were detected in the second allele. The g.1181A>G mutation generated an alternative splicing site in the BCS1L transcript, causing a 19-nucleotides deletion in its 5'UTR region. Decreased BCS1L mRNA and protein levels, and a respiratory chain complex III assembly impairment, determine a pathogenic role for the novel BCS1L mutations.
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http://dx.doi.org/10.1016/j.mito.2009.04.001DOI Listing
September 2009

Infantile mitochondrial encephalomyopathy with unusual phenotype caused by a novel BCS1L mutation in an isolated complex III-deficient patient.

Neuromuscul Disord 2009 Feb 21;19(2):143-6. Epub 2009 Jan 21.

Centro de Investigación, Hospital Universitario 12 de Octubre, Madrid, Spain.

Mutations in BCS1L, a respiratory chain complex III assembly chaperone, constitute a major cause of mitochondrial complex III deficiency and are associated with GRACILE and Björnstad syndromes. Here we describe a 4-year-old infant with hyperlactacidemia, mild liver dysfunction, hypotonia, growth and psychomotor retardation, dysmorphic features and mitochondrial complex III deficiency. Respiratory chain enzyme activities showed an isolated complex III defect in muscle and fibroblasts. Sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed a novel homozygous BCS1L mutation, c.148A>G, which caused a p.T50A substitution at an evolutionarily conserved BCS1L region. The severity of the complex III enzyme defect correlated with decreased amounts of BCS1L and respiratory chain complex III in the affected tissues. Our findings support a pathogenic role for the novel BCS1L mutation in a patient with a singular clinical phenotype.
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http://dx.doi.org/10.1016/j.nmd.2008.11.016DOI Listing
February 2009

Mitochondrial disorders due to nuclear OXPHOS gene defects.

Adv Exp Med Biol 2009 ;652:85-116

Mitochondrial diseases laboratory, Research Center, 12 de Octubre, University Hospital, Madrid, Spain.

Because of the bi-genomic origin of the OXPHOS system, mitochondrial disease-associated mutations have been found in both mtDNA and nuclear structural genes. In the last years, interest has shifted toward mendelian genetics in mitochondrial disease, not only because the majority of the OXPHOS system subunits are encoded by the nuclear genome, but also because a large number of yet unknown nuclear proteins, such as regulatory proteins and assembly factors, are likely involved in its biogenesis and function. A clinical-genetic classification can be proposed for nuclear defects that affect the biogenesis of the OXPHOS system, as follows: (i) disorders due to nuclear gene defects encoding structural components or assembly factors of the OXPHOS complexes, (ii) disorders due to gene defects in the biogenesis of protein constituents of the OXPHOS system, (iii) disorders due to defects in the biosynthesis of non-protein constituents of the respiratory chain, and (iv) disorders due to gene defects encoding proteins involved in mitochondrial dynamics.
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http://dx.doi.org/10.1007/978-90-481-2813-6_7DOI Listing
April 2010

Abeta accumulation in choroid plexus is associated with mitochondrial-induced apoptosis.

Neurobiol Aging 2010 Sep 5;31(9):1569-81. Epub 2008 Oct 5.

Neuroscience Laboratory, Research Center, Hospital 12 de Octubre, Madrid, Spain.

One of the possible mechanisms involved in beta-amyloid (Abeta)-induced neuronal damage is blood-cerebrospinal fluid barrier dysfunction. Recently, we have demonstrated that Alzheimer patients have an elevated expression of Abeta in the choroid plexus (CP), where it could impair the physiological functions of CP epithelium. We investigated whether these alterations were mediated by mitochondrial dysfunction, a common early pathomechanism in Alzheimer's disease. Our main observations were: high Abeta levels; increased nitric oxide levels; impairment of the activity and assembly of mitochondrial respiratory chain complexes I and IV; and a significant increase in reactive oxygen species and caspase expression in CP epithelial cells treated with Abeta. Our results also demonstrate a direct relationship between Abeta toxicity, increased expression of matrix metalloproteinase-9, and blood-cerebrospinal fluid barrier disruption. We propose a sequence of pathological steps that link Abeta accumulation in CP epithelium with an enhanced nitric oxide production, mitochondrial dysfunction, and up-regulation of matrix metalloproteinase-9, which ultimately lead to cell death, and probably to CSF barrier dysfunction.
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http://dx.doi.org/10.1016/j.neurobiolaging.2008.08.017DOI Listing
September 2010

Mitochondrial DNA background modulates the assembly kinetics of OXPHOS complexes in a cellular model of mitochondrial disease.

Hum Mol Genet 2008 Dec 19;17(24):4001-11. Epub 2008 Sep 19.

CIBERER-U723, Hospital Universitario 12 de Octubre, Madrid 28041, Spain.

Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disorder, is mostly due to three mitochondrial DNA (mtDNA) mutations in respiratory chain complex I subunit genes: 3460/ND1, 11778/ND4 and 14484/ND6. Despite considerable clinical evidences, a genetic modifying role of the mtDNA haplogroup background in the clinical expression of LHON remains experimentally unproven. We investigated the effect of mtDNA haplogroups on the assembly of oxidative phosphorylation (OXPHOS) complexes in transmitochondrial hybrids (cybrids) harboring the three common LHON mutations. The steady-state levels of respiratory chain complexes appeared normal in mutant cybrids. However, an accumulation of low molecular weight subcomplexes suggested a complex I assembly/stability defect, which was further demonstrated by reversibly inhibiting mitochondrial protein translation with doxycycline. Our results showed differentially delayed assembly rates of respiratory chain complexes I, III and IV amongst mutants belonging to different mtDNA haplogroups, revealing that specific mtDNA polymorphisms may modify the pathogenic potential of LHON mutations by affecting the overall assembly kinetics of OXPHOS complexes.
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http://dx.doi.org/10.1093/hmg/ddn303DOI Listing
December 2008