29 results on '"Erika Fernandez-Vizarra"'
Search Results
2. Mutation in the MICOS subunit gene APOO (MIC26) associated with an X-linked recessive mitochondrial myopathy, lactic acidosis, cognitive impairment and autistic features
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Alexander J. Whitworth, Julien Prudent, Enrico Baruffini, Anna Yeates, Daniel Almeida do Valle, Alan J. Robinson, Erika Fernandez-Vizarra, Michele Brischigliaro, Aurelio Reyes, Mara Lúcia Schmitz Ferreira Santos, Ricardo L.R. Souza, Mark H. Johnson, Massimo Zeviani, Bruno Augusto Telles, Andrea Degiorgi, Cristiane Benincá, Vanessa Zanette, Prudent, Julien [0000-0003-3821-6088], Whitworth, Alex [0000-0002-1154-6629], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Saccharomyces cerevisiae Proteins ,clinical genetics ,genetics ,metabolic disorders ,neuromuscular disease ,Acidosis, Lactic ,Animals ,Apolipoproteins ,Autistic Disorder ,Cognitive Dysfunction ,Drosophila melanogaster ,Fibroblasts ,Genetic Diseases, X-Linked ,Humans ,Membrane Proteins ,Mitochondrial Membranes ,Mitochondrial Myopathies ,Mitochondrial Proteins ,Protein Binding ,Saccharomyces cerevisiae ,Biology ,Mitochondrion ,medicine.disease_cause ,03 medical and health sciences ,0302 clinical medicine ,Mitochondrial myopathy ,Genetics ,medicine ,Inner mitochondrial membrane ,Genetics (clinical) ,X-linked recessive inheritance ,Exome sequencing ,Mutation ,Lactic ,X-Linked ,medicine.disease ,biology.organism_classification ,030104 developmental biology ,Genetic Diseases ,Lactic acidosis ,Acidosis ,030217 neurology & neurosurgery - Abstract
BackgroundMitochondria provide ATP through the process of oxidative phosphorylation, physically located in the inner mitochondrial membrane (IMM). The mitochondrial contact site and organising system (MICOS) complex is known as the ‘mitoskeleton’ due to its role in maintaining IMM architecture. APOO encodes MIC26, a component of MICOS, whose exact function in its maintenance or assembly has still not been completely elucidated.MethodsWe have studied a family in which the most affected subject presented progressive developmental delay, lactic acidosis, muscle weakness, hypotonia, weight loss, gastrointestinal and body temperature dysautonomia, repetitive infections, cognitive impairment and autistic behaviour. Other family members showed variable phenotype presentation. Whole exome sequencing was used to screen for pathological variants. Patient-derived skin fibroblasts were used to confirm the pathogenicity of the variant found in APOO. Knockout models in Drosophila melanogaster and Saccharomyces cerevisiae were employed to validate MIC26 involvement in MICOS assembly and mitochondrial function.ResultsA likely pathogenic c.350T>C transition was found in APOO predicting an I117T substitution in MIC26. The mutation caused impaired processing of the protein during import and faulty insertion into the IMM. This was associated with altered MICOS assembly and cristae junction disruption. The corresponding mutation in MIC26 or complete loss was associated with mitochondrial structural and functional deficiencies in yeast and D. melanogaster models.ConclusionThis is the first case of pathogenic mutation in APOO, causing altered MICOS assembly and neuromuscular impairment. MIC26 is involved in the assembly or stability of MICOS in humans, yeast and flies.
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- 2020
3. CG7630 is the Drosophila melanogaster homolog of the cytochrome c oxidase subunit COX7B
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Michele Brischigliaro, Alfredo Cabrera‐Orefice, Mattia Sturlese, Dei M Elurbe, Elena Frigo, Erika Fernandez‐Vizarra, Stefano Moro, Martijn A Huynen, Susanne Arnold, Carlo Viscomi, and Massimo Zeviani
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Mammals ,Proteomics ,D. melanogaster ,respiratory chain ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Biochemistry ,COX7B ,Mitochondria ,Electron Transport Complex IV ,Drosophila melanogaster ,cytochrome c oxidase ,mitochondria ,Amino Acid Sequence ,Animals ,Genetics ,Molecular Biology - Abstract
Contains fulltext : 251162.pdf (Publisher’s version ) (Open Access) The mitochondrial respiratory chain (MRC) is composed of four multiheteromeric enzyme complexes. According to the endosymbiotic origin of mitochondria, eukaryotic MRC derives from ancestral proteobacterial respiratory structures consisting of a minimal set of complexes formed by a few subunits associated with redox prosthetic groups. These enzymes, which are the "core" redox centers of respiration, acquired additional subunits, and increased their complexity throughout evolution. Cytochrome c oxidase (COX), the terminal component of MRC, has a highly interspecific heterogeneous composition. Mammalian COX consists of 14 different polypeptides, of which COX7B is considered the evolutionarily youngest subunit. We applied proteomic, biochemical, and genetic approaches to investigate the COX composition in the invertebrate model Drosophila melanogaster. We identified and characterized a novel subunit which is widely different in amino acid sequence, but similar in secondary and tertiary structures to COX7B, and provided evidence that this object is in fact replacing the latter subunit in virtually all protostome invertebrates. These results demonstrate that although individual structures may differ the composition of COX is functionally conserved between vertebrate and invertebrate species.
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- 2022
4. Cooperative assembly of the mitochondrial respiratory chain
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Cristina Ugalde and Erika Fernandez-Vizarra
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Electron Transport ,Mammals ,assembly factors ,mitochondria ,supercomplexes ,Mitochondrial Membranes ,cooperative assembly model ,plasticity model ,respiratory chain organization ,Animals ,Molecular Biology ,Biochemistry - Abstract
Deep understanding of the pathophysiological role of the mitochondrial respiratory chain (MRC) relies on a well-grounded model explaining how its biogenesis is regulated. The lack of a consistent framework to clarify the modes and mechanisms governing the assembly of the MRC complexes and supercomplexes (SCs) works against progress in the field. The plasticity model was postulated as an attempt to explain the coexistence of mammalian MRC complexes as individual entities and associated in SC species. However, mounting data accumulated throughout the years question the universal validity of the plasticity model as originally proposed. Instead, as we argue here, a cooperative assembly model provides a much better explanation to the phenomena observed when studying MRC biogenesis in physiological and pathological settings.
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- 2022
5. Measurement of mitochondrial respiratory chain enzymatic activities in Drosophila melanogaster samples
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Michele Brischigliaro, Elena Frigo, Erika Fernandez-Vizarra, Paolo Bernardi, and Carlo Viscomi
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General Immunology and Microbiology ,General Neuroscience ,Citrate (si)-Synthase ,General Biochemistry, Genetics and Molecular Biology ,Mitochondria ,Electron Transport ,Metabolism ,Model Organisms ,Drosophila melanogaster ,Protein Biochemistry ,Mitochondrial Membranes ,Cell separation/fractionation ,Animals - Published
- 2022
6. Mitochondrial disorders of the OXPHOS system
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Massimo Zeviani and Erika Fernandez-Vizarra
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biogenesis of the respiratory chain ,Mitochondrial Diseases ,Cellular respiration ,Mitochondrial disease ,Biophysics ,oxidative phosphorylation ,mitochondrial respiratory chain ,Oxidative phosphorylation ,mitochondrial proton pumping ,DNA, Mitochondrial ,Biochemistry ,Electron Transport ,03 medical and health sciences ,Adenosine Triphosphate ,Structural Biology ,ATP production ,Mitochondrial respiratory chain ,mitochondrial disease ,mitochondrial electrochemical gradient ,mitochondrial potential ,respiratory complex ,respiratory supercomplex ,Genetics ,medicine ,Animals ,Humans ,Electron Transport Chain Complex Proteins ,Mitochondria ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,ATP synthase ,biology ,Chemiosmosis ,Chemistry ,030302 biochemistry & molecular biology ,Cell Biology ,DNA ,medicine.disease ,Electron transport chain ,Cell biology ,Mitochondrial ,biology.protein ,Biogenesis - Abstract
Mitochondrial disorders are among the most frequent inborn errors of metabolism, their primary cause being the dysfunction of the oxidative phosphorylation system (OXPHOS). OXPHOS is composed of the electron transport chain (ETC), formed by four multimeric enzymes and two mobile electron carriers, plus an ATP synthase [also called complex V (cV)]. The ETC performs the redox reactions involved in cellular respiration while generating the proton motive force used by cV to synthesize ATP. OXPHOS biogenesis involves multiple steps, starting from the expression of genes encoded in physically separated genomes, namely the mitochondrial and nuclear DNA, to the coordinated assembly of components and cofactors building each individual complex and eventually the supercomplexes. The genetic cause underlying around half of the diagnosed mitochondrial disease cases is currently known. Many of these cases result from pathogenic variants in genes encoding structural subunits or additional factors directly involved in the assembly of the ETC complexes. Here, we review the historical and most recent findings concerning the clinical phenotypes and the molecular pathological mechanisms underlying this particular group of disorders.
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- 2021
7. Neural stem cells traffic functional mitochondria via extracellular vesicles
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Erika Fernandez-Vizarra, Carlo Viscomi, Ágnes Kittel, Sisareuth Tan, Christian Frezza, Stefano Pluchino, Cristiane Benincá, Nunzio Vicario, Massimo Zeviani, Tommaso Leonardi, Ben Peacock, Paul J. Lehner, Alice Braga, Joshua D. Bernstock, Aletta Van Den Bosch, Jayden A. Smith, Karin H. Muller, Carlos Bastos, Edit I. Buzás, Luca Peruzzotti-Jametti, Giulia Manferrari, Cory M. Willis, Alain Brisson, Nunzio Iraci, Rebecca Rogall, Nuno Faria, Grzegorz Krzak, James C Williamson, Nicholas J Matheson, Iacopo Bicci, Chimie et Biologie des Membranes et des Nanoobjets (CBMN), École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), European Project: 258803, Peruzzotti-Jametti, Luca [0000-0002-9396-5607], Bernstock, Joshua D [0000-0002-7814-3867], Willis, Cory M [0000-0001-7938-7276], Manferrari, Giulia [0000-0001-7062-1142], Rogall, Rebecca [0000-0003-0605-2322], Fernandez-Vizarra, Erika [0000-0002-2469-142X], Williamson, James C [0000-0002-2009-189X], Braga, Alice [0000-0003-3273-9742], van den Bosch, Aletta [0000-0001-8886-8928], Leonardi, Tommaso [0000-0002-4449-1863], Benincá, Cristiane [0000-0001-7933-860X], Vicario, Nunzio [0000-0001-5934-3962], Tan, Sisareuth [0000-0003-3633-6318], Bicci, Iacopo [0000-0001-6994-3857], Iraci, Nunzio [0000-0003-2146-9329], Smith, Jayden A [0000-0003-2307-8452], Peacock, Ben [0000-0002-7823-8719], Muller, Karin H [0000-0003-4693-8558], Brisson, Alain [0000-0003-0342-352X], Matheson, Nicholas J [0000-0002-3318-1851], Apollo - University of Cambridge Repository, Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Bernstock, Joshua D. [0000-0002-7814-3867], Willis, Cory M. [0000-0001-7938-7276], Williamson, James C. [0000-0002-2009-189X], Smith, Jayden A. [0000-0003-2307-8452], Muller, Karin H. [0000-0003-4693-8558], and Matheson, Nicholas J. [0000-0002-3318-1851]
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0301 basic medicine ,Central Nervous System ,Proteomics ,[SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology ,Cell Membranes ,Artificial Gene Amplification and Extension ,Mitochondrion ,Outer membrane proteins ,Inbred C57BL ,Exosomes ,Biochemistry ,Nervous System ,Transgenic ,White Blood Cells ,Database and Informatics Methods ,Mice ,0302 clinical medicine ,Neural Stem Cells ,Animal Cells ,Medicine and Health Sciences ,Computer software ,Biology (General) ,Energy-Producing Organelles ,Cells, Cultured ,Phagocytes ,Cultured ,Proteomic Databases ,General Neuroscience ,Neural stem cell ,Cell biology ,Mitochondria ,Polymerase chain reaction ,medicine.anatomical_structure ,Female ,Stem cell ,Cellular Structures and Organelles ,Cellular Types ,Anatomy ,General Agricultural and Biological Sciences ,Research Article ,QH301-705.5 ,Cells ,Immune Cells ,Central nervous system ,Immunology ,Green Fluorescent Proteins ,Mice, Transgenic ,Biology ,Bioenergetics ,Research and Analysis Methods ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Extracellular Vesicles ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Organelle ,medicine ,Animals ,Vesicles ,Molecular Biology Techniques ,Molecular Biology ,Blood Cells ,General Immunology and Microbiology ,Biology and Life Sciences ,Membrane Proteins ,Biological Transport ,Mesenchymal Stem Cells ,Cell Biology ,Microvesicles ,Transplantation ,Mice, Inbred C57BL ,030104 developmental biology ,Biological Databases ,030217 neurology & neurosurgery - Abstract
Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases., This study shows that neural stem cells are able to transfer functional mitochondria via extracellular vesicles to target cells both in vitro and in vivo, suggesting that functional mitochondrial transfer via extracellular vesicles is a signaling mechanism used by neural stem cells to modulate the physiology and metabolism of target cells.
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- 2021
8. NDUFS3 depletion permits complex I maturation and reveals TMEM126A/OPA7 as an assembly factor binding the ND4-module intermediate
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Anna Maria Porcelli, Giuseppe Gasparre, Ivana Kurelac, Erika Fernandez-Vizarra, Luigi D'Angelo, Shujing Ding, Nikkitha Umesh-Ganesh, Monica De Luise, Massimo Zeviani, Elisa Astro, Luisa Iommarini, Ian M. Fearnley, D’Angelo, Luigi, Astro, Elisa, De Luise, Monica, Kurelac, Ivana, Umesh-Ganesh, Nikkitha, Ding, Shujing, Fearnley, Ian M., Gasparre, Giuseppe, Zeviani, Massimo, Porcelli, Anna Maria, Fernandez-Vizarra, Erika, and Iommarini, Luisa
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Models, Molecular ,Proteomics ,0301 basic medicine ,Bioenergetics ,Protein Conformation ,NDUFS3 ,SILAC ,Gene Knockout Techniques ,Mice ,0302 clinical medicine ,Models ,Stable isotope labeling by amino acids in cell culture ,Biology (General) ,Gene Editing ,Disease gene ,Tumor ,Chemistry ,CI ,optic atrophy type 7 ,respiratory complex I CI NDUFS3 CI modules assembly factor TMEM126A SILAC optic atrophy type 7 ,Mitochondria ,Cell biology ,CI modules ,TMEM126A ,assembly factor ,respiratory complex I ,Animals ,Binding Sites ,CRISPR-Cas Systems ,Cell Line, Tumor ,Electron Transport Complex I ,Gene Expression Regulation ,HCT116 Cells ,Humans ,Melanocytes ,Membrane Proteins ,Mitochondrial Membranes ,NADH Dehydrogenase ,Optic Atrophy ,Osteoblasts ,Protein Binding ,Mitochondrial respiratory chain ,QH301-705.5 ,Protein subunit ,Mitochondrial disease ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,03 medical and health sciences ,medicine ,Molecular ,medicine.disease ,030104 developmental biology ,030217 neurology & neurosurgery ,Function (biology) ,Biogenesis - Abstract
Summary Complex I (CI) is the largest enzyme of the mitochondrial respiratory chain, and its defects are the main cause of mitochondrial disease. To understand the mechanisms regulating the extremely intricate biogenesis of this fundamental bioenergetic machine, we analyze the structural and functional consequences of the ablation of NDUFS3, a non-catalytic core subunit. We show that, in diverse mammalian cell types, a small amount of functional CI can still be detected in the complete absence of NDUFS3. In addition, we determine the dynamics of CI disassembly when the amount of NDUFS3 is gradually decreased. The process of degradation of the complex occurs in a hierarchical and modular fashion in which the ND4 module remains stable and bound to TMEM126A. We, thus, uncover the function of TMEM126A, the product of a disease gene causing recessive optic atrophy as a factor necessary for the correct assembly and function of CI., Graphical abstract, Highlights • A fraction of functional complex I assembles without the core subunit NDUFS3 • Complex I disassembly differentially affects its submodules • With NDUFS3 absent, the ND4-module of the P-distal domain remains mostly stable • The TMEM126A/OPA7 factor interacts with the ND4-module and is necessary for CI maturation, D’Angelo et al. show that eliminating NDUFS3 does not completely abolish respiratory complex I maturation. Differential degradation of complex I subunits belonging to different structural/functional modules is triggered by NDUFS3 repression. The ND4 module remains stable and is bound to TMEM126A, which is, here, identified as a complex I assembly factor.
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- 2021
9. Mutation in the MICOS subunit gene
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Cristiane, Benincá, Vanessa, Zanette, Michele, Brischigliaro, Mark, Johnson, Aurelio, Reyes, Daniel Almeida do, Valle, Alan, J Robinson, Andrea, Degiorgi, Anna, Yeates, Bruno Augusto, Telles, Julien, Prudent, Enrico, Baruffini, Mara Lucia, S F Santos, Ricardo Lehtonen, R de Souza, Erika, Fernandez-Vizarra, Alexander J, Whitworth, and Massimo, Zeviani
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Saccharomyces cerevisiae Proteins ,Membrane Proteins ,Mitochondrial Myopathies ,Genetic Diseases, X-Linked ,Saccharomyces cerevisiae ,Fibroblasts ,Article ,Mitochondrial Proteins ,Apolipoproteins ,Drosophila melanogaster ,Mitochondrial Membranes ,Animals ,Humans ,Acidosis, Lactic ,Cognitive Dysfunction ,Autistic Disorder ,Protein Binding - Abstract
Background Mitochondria provide ATP through the process of oxidative phosphorylation, physically located in the inner mitochondrial membrane (IMM). The mitochondrial contact site and organising system (MICOS) complex is known as the ’mitoskeleton’ due to its role in maintaining IMM architecture. APOO encodes MIC26, a component of MICOS, whose exact function in its maintenance or assembly has still not been completely elucidated. Methods We have studied a family in which the most affected subject presented progressive developmental delay, lactic acidosis, muscle weakness, hypotonia, weight loss, gastrointestinal and body temperature dysautonomia, repetitive infections, cognitive impairment and autistic behaviour. Other family members showed variable phenotype presentation. Whole exome sequencing was used to screen for pathological variants. Patient-derived skin fibroblasts were used to confirm the pathogenicity of the variant found in APOO. Knockout models in Drosophila melanogaster and Saccharomyces cerevisiae were employed to validate MIC26 involvement in MICOS assembly and mitochondrial function. Results A likely pathogenic c.350T>C transition was found in APOO predicting an I117T substitution in MIC26. The mutation caused impaired processing of the protein during import and faulty insertion into the IMM. This was associated with altered MICOS assembly and cristae junction disruption. The corresponding mutation in MIC26 or complete loss was associated with mitochondrial structural and functional deficiencies in yeast and D. melanogaster models. Conclusion This is the first case of pathogenic mutation in APOO, causing altered MICOS assembly and neuromuscular impairment. MIC26 is involved in the assembly or stability of MICOS in humans, yeast and flies.
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- 2020
10. A homozygous MRPL24 mutation causes a complex movement disorder and affects the mitoribosome assembly
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Michal Minczuk, Romina Oliva, Anna Ardissone, Federica Morani, Maria Marchese, Daniela Verrigni, Filippo M. Santorelli, Daniele Ghezzi, Teresa Rizza, Claudia Nesti, Giulia Trani, Alessandra Torraco, Massimo Zeviani, Christian Daniel Mutti, Gessica Vasco, Erika Fernandez-Vizarra, Rosalba Carrozzo, Michela Di Nottia, and Enrico Bertini
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0301 basic medicine ,Male ,Ribosomal Proteins ,Mitochondrial translation ,Protein subunit ,Mutant ,MRPL24 ,Mitochondrial disorders ,Mitochondrial protein synthesis ,Mitoribosomes ,Molecular modeling ,Movement disorder ,Protein interactions ,Zebrafish ,Animals ,Cerebellum ,Female ,Humans ,Infant ,Leviviridae ,Mitochondrial Proteins ,Movement Disorders ,Quadriceps Muscle ,Gene mutation ,lcsh:RC321-571 ,03 medical and health sciences ,0302 clinical medicine ,Ribosomal protein ,Mitochondrial ribosome ,Missense mutation ,Mitochondrial respiratory chain complex I ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Chemistry ,Cell biology ,030104 developmental biology ,Neurology ,030217 neurology & neurosurgery - Abstract
Mitochondrial ribosomal protein large 24 (MRPL24) is 1 of the 82 protein components of mitochondrial ribosomes, playing an essential role in the mitochondrial translation process. We report here on a baby girl with cerebellar atrophy, choreoathetosis of limbs and face, intellectual disability and a combined defect of complexes I and IV in muscle biopsy, caused by a homozygous missense mutation identified in MRPL24. The variant predicts a Leu91Pro substitution at an evolutionarily conserved site. Using human mutant cells and the zebrafish model, we demonstrated the pathological role of the identified variant. In fact, in fibroblasts we observed a significant reduction of MRPL24 protein and of mitochondrial respiratory chain complex I and IV subunits, as well a markedly reduced synthesis of the mtDNA-encoded peptides. In zebrafish we demonstrated that the orthologue gene is expressed in metabolically active tissues, and that gene knockdown induced locomotion impairment, structural defects and low ATP production. The motor phenotype was complemented by human WT but not mutant cRNA. Moreover, sucrose density gradient fractionation showed perturbed assembly of large subunit mitoribosomal proteins, suggesting that the mutation leads to a conformational change in MRPL24, which is expected to cause an aberrant interaction of the protein with other components of the 39S mitoribosomal subunit.
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- 2020
11. Assembly of mammalian oxidative phosphorylation complexes I–V and supercomplexes
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Alba Signes, Erika Fernandez-Vizarra, Fernandez-Vizarra, Erika [0000-0002-2469-142X], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Mitochondrial disease ,oxidative phosphorylation ,Oxidative phosphorylation ,Review Article ,Mitochondrion ,Biochemistry ,Cofactor ,Electron Transport ,03 medical and health sciences ,Multienzyme Complexes ,medicine ,atp synthase ,Animals ,Humans ,Inner mitochondrial membrane ,Molecular Biology ,Review Articles ,chemistry.chemical_classification ,Mammals ,biology ,ATP synthase ,Chemistry ,electron transport chain ,medicine.disease ,Electron transport chain ,Cell biology ,Mitochondria ,030104 developmental biology ,Enzyme ,mitochondria ,respiratory chain complex assembly ,Oxidative Phosphorylation ,biology.protein - Abstract
The assembly of the five oxidative phosphorylation system (OXPHOS) complexes in the inner mitochondrial membrane is an intricate process. The human enzymes comprise core proteins, performing the catalytic activities, and a large number of ‘supernumerary’ subunits that play essential roles in assembly, regulation and stability. The correct addition of prosthetic groups as well as chaperoning and incorporation of the structural components require a large number of factors, many of which have been found mutated in cases of mitochondrial disease. Nowadays, the mechanisms of assembly for each of the individual complexes are almost completely understood and the knowledge about the assembly factors involved is constantly increasing. On the other hand, it is now well established that complexes I, III and IV interact with each other, forming the so-called respiratory supercomplexes or ‘respirasomes’, although the pathways that lead to their formation are still not completely clear. This review is a summary of our current knowledge concerning the assembly of complexes I–V and of the supercomplexes.
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- 2018
12. APOPT1/COA8 assists COX assembly and is oppositely regulated by UPS and ROS
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Elizabeth C. Hinchy, Raffaele Cerutti, Rosalba Carrozzo, James A. Nathan, Erika Fernandez-Vizarra, Michael P. Murphy, Carlo Viscomi, Alba Signes, Daniele Ghezzi, Cristiane Benincá, Massimo Zeviani, Anna S Dickson, Enrico Bertini, Ghezzi, Daniele [0000-0002-9358-1566], Bertini, Enrico [0000-0001-9276-4590], Nathan, James A [0000-0002-0248-1632], Viscomi, Carlo [0000-0001-6050-0566], Fernandez-Vizarra, Erika [0000-0002-2469-142X], Zeviani, Massimo [0000-0002-9067-5508], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Medicine (General) ,APOPT1-COA8 ,QH426-470 ,medicine.disease_cause ,APOPT1‐COA8 ,Mice ,0302 clinical medicine ,cytochrome c oxidase ,Cells, Cultured ,Research Articles ,chemistry.chemical_classification ,Mice, Knockout ,reactive oxygen species ,Oxidase test ,mitochondrial encephalopathy ,proteasome–ubiquitin system ,Animals ,Apoptosis Regulatory Proteins ,Electron Transport Complex IV ,Genetic Complementation Test ,Humans ,Mitochondrial Proteins ,Reactive Oxygen Species ,Protein Multimerization ,Unfolded Protein Response ,Cultured ,biology ,Chemistry ,Cell biology ,Knockout mouse ,Molecular Medicine ,Research Article ,Cells ,Knockout ,03 medical and health sciences ,R5-920 ,Genetics ,medicine ,Cytochrome c oxidase ,Gene ,Reactive oxygen species ,030104 developmental biology ,Apoptosis ,biology.protein ,Genetics, Gene Therapy & Genetic Disease ,030217 neurology & neurosurgery ,Function (biology) ,Oxidative stress - Abstract
Loss‐of‐function mutations in APOPT1, a gene exclusively found in higher eukaryotes, cause a characteristic type of cavitating leukoencephalopathy associated with mitochondrial cytochrome c oxidase (COX) deficiency. Although the genetic association of APOPT1 pathogenic variants with isolated COX defects is now clear, the biochemical link between APOPT1 function and COX has remained elusive. We investigated the molecular role of APOPT1 using different approaches. First, we generated an Apopt1 knockout mouse model which shows impaired motor skills, e.g., decreased motor coordination and endurance, associated with reduced COX activity and levels in multiple tissues. In addition, by achieving stable expression of wild‐type APOPT1 in control and patient‐derived cultured cells we ruled out a role of this protein in apoptosis and established instead that this protein is necessary for proper COX assembly and function. On the other hand, APOPT1 steady‐state levels were shown to be controlled by the ubiquitination–proteasome system (UPS). Conversely, in conditions of increased oxidative stress, APOPT1 is stabilized, increasing its mature intramitochondrial form and thereby protecting COX from oxidatively induced degradation.
- Published
- 2018
13. Bioenergetic consequences from xenotopic expression of a tunicate AOX in mouse mitochondria: Switch from RET and ROS to FET
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Marten Szibor, T. M. Gainutdinov, Erika Fernandez-Vizarra, Ilka Wittig, Grazyna Debska-Vielhaber, Frank N. Gellerich, Juliana Heidler, Eric Dufour, Anthony L. Moore, Carlo Viscomi, Zemfira Gizatullina, Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology, Tampere University, Viscomi, Carlo [0000-0001-6050-0566], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Alternative oxidase ,Biolääketieteet - Biomedicine ,Citric Acid Cycle ,Biophysics ,Respiratory chain ,Gene Expression ,Oxidative phosphorylation ,Biochemistry ,Mitochondria, Heart ,Electron Transport ,Mice ,03 medical and health sciences ,Oxygen Consumption ,0302 clinical medicine ,Xenotopic expression ,Alternative oxidase (AOX) ,Mitochondria ,OXPHOS ,Quinone pool ,ROS ,Aldehyde Oxidase ,Animals ,Ciona intestinalis ,Electron Transport Complex I ,Reactive Oxygen Species ,Succinate Dehydrogenase ,biology ,Chemistry ,Succinate dehydrogenase ,Heart ,Cell Biology ,Electron transport chain ,Reverse electron flow ,Cell biology ,030104 developmental biology ,030220 oncology & carcinogenesis ,Coenzyme Q – cytochrome c reductase ,biology.protein - Abstract
Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc1 complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.
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- 2020
14. SURF1 knockout cloned pigs: Early onset of a severe lethal phenotype
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C. Corona, Emanuela Bottani, P. Crociara, I. Di Meo, Mark A. Johnson, Carlo Viscomi, Valeria Tiranti, S. Grifoni, Cesare Galli, Erika Fernandez-Vizarra, C. Casalone, Corinne Quadalti, Giovanna Lazzari, Irina Lagutina, Dario Brunetti, Massimo Zeviani, Raffaele Cerutti, Andrea Perota, Roberto Duchi, and Alan J. Robinson
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0301 basic medicine ,Central Nervous System ,Male ,Nuclear Transfer Techniques ,Sus scrofa ,Mitochondrion ,Animals, Genetically Modified ,SURF1 KO ,Gene Knockout Techniques ,0302 clinical medicine ,Genome editing ,Leigh syndrome ,Mitochondrial disease ,Pig ,Animals ,Animals, Newborn ,Behavior, Animal ,CRISPR-Cas Systems ,Cells, Cultured ,Down-Regulation ,Electron Transport Complex IV ,Female ,Fibroblasts ,Gene Editing ,Humans ,Jejunum ,Leigh Disease ,Membrane Proteins ,Mitochondria ,Mitochondrial Proteins ,Muscle, Skeletal ,Primary Cell Culture ,Disease Models, Animal ,SURF1 ,Cultured ,Skeletal ,Phenotype ,Muscle ,Molecular Medicine ,Cells ,Genetically Modified ,Biology ,Article ,Andrology ,03 medical and health sciences ,medicine ,Leigh disease ,Molecular Biology ,Gene ,Behavior ,Animal ,Wild type ,Newborn ,medicine.disease ,030104 developmental biology ,MRNA Sequencing ,Disease Models ,030217 neurology & neurosurgery - Abstract
Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF1−/− mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1−/− and SURF1−/+ pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1−/− pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1−/+ and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1−/− tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down-regulated in SURF1−/− skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1−/− piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics., Highlights • The full sequence of pig SURF1 gene was determined. • SURF1 gene was disrupted in pig by gene editing and somatic cell nuclear transfer. • SURF1−/− piglets showed an early onset lethal phenotype. • Mitochondrial bioenergetics was impaired in the skeletal muscle of SURF1−/− pigs.
- Published
- 2017
15. TTC19 Plays a Husbandry Role on UQCRFS1 Turnover in the Biogenesis of Mitochondrial Respiratory Complex III
- Author
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Raffaele Cerutti, Carla Giordano, Sabrina Ravaglia, Giulia d'Amati, Erika Fernandez-Vizarra, Massimo Zeviani, Carlo Viscomi, Sukru Anil Dogan, Ian M. Fearnley, Michael E. Harbour, and Emanuela Bottani
- Subjects
0301 basic medicine ,Iron-Sulfur Proteins ,Male ,Mitochondrial Diseases ,Dimer ,Inbred C57BL ,Nervous System ,chemistry.chemical_compound ,Electron Transport Complex III ,Mice ,complex III deficiency ,mitochondrial complex III ,mitochondrial disease ,mitochondrial quality control ,mitochondrial respiratory chain ,mouse model ,Rieske protein ,TTC19 ,UQCRFS1 ,Animals ,Behavior, Animal ,Disease Models, Animal ,Female ,Genotype ,HeLa Cells ,Humans ,Kinetics ,Membrane Proteins ,Mice, Inbred C57BL ,Mice, Knockout ,Mitochondria ,Mitochondrial Proteins ,Motor Activity ,Nerve Degeneration ,Phenotype ,Protein Binding ,Protein Stability ,Proteolysis ,Reactive Oxygen Species ,Respiratory system ,chemistry.chemical_classification ,biology ,Biochemistry ,Knockout ,03 medical and health sciences ,Molecular Biology ,Reactive oxygen species ,Behavior ,Animal ,Cell Biology ,Mitochondrial respiratory chain complex III ,030104 developmental biology ,chemistry ,Coenzyme Q – cytochrome c reductase ,Disease Models ,biology.protein ,rieske protein ,Biogenesis - Abstract
Loss-of-function mutations in TTC19 (tetra-tricopeptide repeat domain 19) have been associated with severe neurological phenotypes and mitochondrial respiratory chain complex III deficiency. We previously demonstrated the mitochondrial localization of TTC19 and its link with complex III biogenesis. Here we provide detailed insight into the mechanistic role of TTC19, by investigating a Ttc19?/? mouse model that shows progressive neurological and metabolic decline, decreased complex III activity, and increased production of reactive oxygen species. By using both the Ttc19?/? mouse model and a range of human cell lines, we demonstrate that TTC19 binds to the fully assembled complex III dimer, i.e., after the incorporation of the iron-sulfur Rieske protein (UQCRFS1). The in situ maturation of UQCRFS1 produces N-terminal polypeptides, which remain bound to holocomplex III. We show that, in normal conditions, these UQCRFS1 fragments are rapidly removed, but when TTC19 is absent they accumulate within complex III, causing its structural and functional impairment.
- Published
- 2017
16. COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation
- Author
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Susana Cadenas, Inés García-Consuegra, Ana Bratic, Nils-Göran Larsson, Joaquín Arenas, Cristina Ugalde, Aitor Delmiro, Arnaud Mourier, Teresa Lobo-Jarne, Rafael Pérez-Pérez, Erika Fernandez-Vizarra, Dusanka Milenkovic, Miguel A. Martín, Alberto García-Bartolomé, Swedish Research Council, European Research Council, Association Française contre les Myopathies, Comunidad de Madrid, Instituto de Salud Carlos III, and German Research Foundation
- Subjects
0301 basic medicine ,Respiratory chain ,Gene Expression ,Oxidative phosphorylation ,Plasma protein binding ,Biology ,Mitochondrion ,General Biochemistry, Genetics and Molecular Biology ,Mitochondria, Heart ,Oxidative Phosphorylation ,Article ,Electron Transport ,Electron Transport Complex IV ,03 medical and health sciences ,Electron Transport Complex III ,Mice ,Animals ,Humans ,lcsh:QH301-705.5 ,Heart metabolism ,Electron Transport Complex I ,Protein Stability ,Myocardium ,Heart ,HEK293 Cells ,HeLa Cells ,Mitochondrial Membranes ,Protein Binding ,Cell biology ,Mitochondria ,030104 developmental biology ,Mitochondrial respiratory chain ,Biochemistry ,lcsh:Biology (General) ,Respirasome ,Biogenesis - Abstract
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 III+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., Instituto de Salud Carlos III (grant numbers PI11-00182 and PI14-00209 to C.U., PI12-01683 to M.A.M., and PI12-00933 to S.C.), by Comunidad Autónoma de Madrid (P2010/BMD-2361 to C.U. and P2010/BMD-2402 to M.A.M. and S.C.), by European FEDER Funds, by Association Française contre les Myopathies (16086) to E.F.V., by an European Research Council advanced investigator grant (268897) and grants from the Deutsche Forschungsgemeinschaft (SFB829) and the Swedish Research Council (2015-00418) to N.G.L., and by NIH-NIGMS (1R01GM105781-01) to C.U.
- Published
- 2016
17. Defective PITRM1 mitochondrial peptidase is associated with Aβ amyloidotic neurodegeneration
- Author
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Dario Brunetti, Helge Boman, Aurelio Reyes, Cristina Dallabona, Raffaele Cerutti, Per M. Knappskog, Pedro Teixeira, Carmela Preziuso, Giulia d'Amati, Carlo Viscomi, Stefan Johansson, Enrico Baruffini, Massimo Zeviani, Janniche Torsvik, Ileana Ferrero, Paweł Sztromwasser, Elzbieta Glaser, Laurence A. Bindoff, Wenche Telstad, Paola Goffrini, Erika Fernandez-Vizarra, Reyes Tellez, Aurelio [0000-0003-2876-2202], Viscomi, Carlo [0000-0001-6050-0566], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,Mice ,Models ,News & Views ,Cognitive decline ,Inner mitochondrial membrane ,2. Zero hunger ,Genetics ,Histocytochemistry ,Amyloid beta ,Mitochondrial disease ,Mitochondrial targeting sequence ,Neurodegeneration ,Pitrilysin 1 ,Amyloid beta-Peptides ,Animals ,Brain ,Disease Models, Animal ,Humans ,Magnetic Resonance Imaging ,Metalloendopeptidases ,Models, Biological ,Muscle, Skeletal ,Mutant Proteins ,Mutation, Missense ,Neurodegenerative Diseases ,Saccharomyces cerevisiae ,Siblings ,Skeletal ,3. Good health ,Spinocerebellar ataxia ,Molecular Medicine ,Muscle ,amyloid beta ,mitochondrial disease ,mitochondrial targeting sequence ,neurodegeneration ,pitrilysin 1 ,Biology ,03 medical and health sciences ,medicine ,Animal ,medicine.disease ,Biological ,Molecular biology ,030104 developmental biology ,Proteostasis ,Metabolism ,Disease Models ,Mutation ,biology.protein ,DNAJA3 ,Genetics, Gene Therapy & Genetic Disease ,Missense ,Neuroscience - Abstract
Mitochondrial dysfunction and altered proteostasis are central features of neurodegenerative diseases. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests oligopeptides, including the mitochondrial targeting sequences that are cleaved from proteins imported across the inner mitochondrial membrane and the mitochondrial fraction of amyloid beta (Aβ). We identified two siblings carrying a homozygous PITRM1 missense mutation (c.548G>A, p.Arg183Gln) associated with an autosomal recessive, slowly progressive syndrome characterised by mental retardation, spinocerebellar ataxia, cognitive decline and psychosis. The pathogenicity of the mutation was tested in vitro , in mutant fibroblasts and skeletal muscle, and in a yeast model. A Pitrm1 +/− heterozygous mouse showed progressive ataxia associated with brain degenerative lesions, including accumulation of Aβ‐positive amyloid deposits. Our results show that PITRM1 is responsible for significant Aβ degradation and that impairment of its activity results in Aβ accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.
- Published
- 2015
18. Mitochondrial gene expression is regulated at multiple levels and differentially in the heart and liver by thyroid hormones
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José Antonio Enríquez, Acisclo Pérez-Martos, Patricio Fernández-Silva, Julio Montoya, and Erika Fernandez-Vizarra
- Subjects
Male ,Thyroid Hormones ,medicine.medical_specialty ,Transcription, Genetic ,RNA, Mitochondrial ,RNA Stability ,Wistar ,translation ,Mitochondria, Liver ,Citrate (si)-Synthase ,In Vitro Techniques ,Mitochondrion ,Biology ,DNA, Mitochondrial ,mtDNA ,transcription ,RNA stability ,in organello ,thyroid hormones ,Animals ,Electron Transport Complex IV ,Gene Expression Regulation ,Hyperthyroidism ,Hypothyroidism ,Mitochondria, Heart ,Organ Specificity ,Oxidative Phosphorylation ,Protein Biosynthesis ,RNA ,Rats ,Rats, Wistar ,Gene dosage ,Genetic ,Internal medicine ,Genetics ,medicine ,Heart metabolism ,Regulation of gene expression ,Thyroid hormone receptor ,Thyroid ,Heart ,DNA ,General Medicine ,Mitochondrial ,Mitochondria ,medicine.anatomical_structure ,Endocrinology ,Liver ,Mitochondrial biogenesis ,Hormone - Abstract
Biogenesis of the oxidative phosphorylation system (OXPHOS) requires the coordinated expression of the nuclear and the mitochondrial genomes. Thyroid hormones play an important role in cell growth and differentiation and are one of the main effectors in mitochondrial biogenesis. To determine how mtDNA expression is regulated, we have investigated the response of two different tissues, the heart and liver, to the thyroid hormone status in vivo and in vitro. We show here that mtDNA expression is a tightly regulated process and that several levels of control can take place simultaneously. In addition, we show that the mechanisms operating in the control of mtDNA expression and their relevance differ between the two tissues, being gene dosage important only in heart while transcription rate and translation efficiency have more weight in liver cells. Another interesting difference is the lack of a direct effect of thyroid hormones on heart mitochondrial transcription.
- Published
- 2008
19. In vitro transcription termination activity of the Drosophila mitochondrial DNA-binding protein DmTTF
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Julio Montoya, Patricio Fernández-Silva, Paola Loguercio Polosa, Maria Nicola Gadaleta, Marina Roberti, Erika Fernandez-Vizarra, Palmiro Cantatore, Stefania Deceglie, and Francesco Bruni
- Subjects
Transcription termination assay ,Transcription, Genetic ,Termination factor ,Biophysics ,DNA-binding protein ,Biochemistry ,Mitochondrial Proteins ,Transcription (biology) ,Animals ,Drosophila Proteins ,Humans ,Molecular Biology ,Polymerase ,Mitochondrial transcription ,General transcription factor ,biology ,RNA ,Cell Biology ,Molecular biology ,Cell biology ,DNA-Binding Proteins ,Drosophila melanogaster ,TAF2 ,biology.protein ,Drosophila ,Transcription factor II D - Abstract
DmTTF is a Drosophila melanogaster mitochondrial DNA-binding protein which binds specifically to two homologous non-coding sequences located at the 30 ends of blocks of genes encoded on opposite strands. In order to test whether this protein acts as transcription termination factor, we assayed the capacity of DmTTF to arrest in vitro the transcription catalyzed by mitochondrial and bacteriophage RNA polymerases. Experiments with human S-100 extracts showed that DmTTF is able to arrest the transcription catalyzed by human mitochondrial RNA polymerase bidirectionally, independently of the orientation of the proteinDNA complex. On the contrary when T3 or T7 RNA polymerases were used, we found that DmTTF prevalently arrests transcription when the DNA-binding site was placed in the reverse orientation with respect to the incoming enzymes. These results demonstrate that DmTTF is a transcription termination factor with a biased polarity and suggest that the DNA-bound protein is structurally asymmetrical, exposing two different faces to RNA polymerases travelling on opposite directions.
- Published
- 2005
20. Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells
- Author
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José Antonio Enríquez, Manuel J. López-Pérez, and Erika Fernandez-Vizarra
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Male ,Mitochondrial DNA ,Cytological Techniques ,DNA footprinting ,Aminoacylation ,Mitochondrion ,Biology ,DNA, Mitochondrial ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Adenosine Triphosphate ,RNA, Transfer ,Organelle ,Animals ,Tissue Distribution ,Rats, Wistar ,Molecular Biology ,Cells, Cultured ,HSPA9 ,RNA ,Mitochondria ,Rats ,Cell biology ,Biochemistry ,chemistry ,Synapses ,DNA ,Protein Binding ,Synaptosomes - Abstract
This article describes a quick basic method adapted for the purification of mammalian mitochondria from different sources. The organelles obtained using this protocol are suitable for the investigation of biogenetic activities such as enzyme activity, mtDNA, mtRNA, mitochondrial protein synthesis, and mitochondrial tRNA aminoacylation. In addition, these mitochondria are capable of efficient protein import and the investigation of mtDNA/protein interactions by DNA footprinting is also possible.
- Published
- 2002
21. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion
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Vamsi K. Mootha, Pietro Strisciuglio, Emmanuelle Sarzi, Claudia Donnini, Massimo Zeviani, Sarah E. Calvo, Valeria Tiranti, Antonella Spinazzola, Erika Fernandez-Vizarra, Agnès Rötig, Carlo Viscomi, Pio D'Adamo, Salvatore DiMauro, René Massimiliano Marsano, Alicia Chan, Iliana Ferrero, Rossella Parini, Hans Weiher, Franco Carrara, Paolo Gasparini, Spinazzola, A, Viscomi, C, FERNANDEZ VIZARRA, E, Carrara, F, D'Adamo, ADAMO PIO, Calvo, S, Marsano, Rm, Donnini, C, Weiher, H, Strisciuglio, P, Parini, R, Sarzi, E, Chan, A, Dimauro, S, Rtig, A, Gasparini, Paolo, Ferrero, I, Mootha, Vk, Tiranti, V, Zeviani, M., Antonella, Spinazzola, Carlo, Viscomi, Erika Fernandez, Vizarra, Franco, Carrara, Pio, D'Adamo, Sarah, Calvo, René Massimiliano, Marsano, Claudia, Donnini, Hans, Weiher, Strisciuglio, Pietro, Rossella, Parini, Emmanuelle, Sarzi, Alicia, Chan, Salvatore, Dimauro, Agnes, Rötig, Paolo, Gasparini, Iliana, Ferrero, Vamsi K., Mootha, Valeria, Tiranti, and Massimo, Zeviani
- Subjects
Male ,MPV17 ,Mitochondrial DNA ,SUCLA2 ,Cells ,Molecular Sequence Data ,Fluorescent Antibody Technique ,Mitochondrion ,Biology ,DGUOK ,DNA, Mitochondrial ,Mitochondrial depletion ,Chromosomes ,Mice ,Genetics ,medicine ,Amino Acid Sequence ,Animals ,Cells, Cultured ,Chromosomes, Human, Pair 2 ,Cloning, Molecular ,Female ,Humans ,Intracellular Membranes ,Liver Diseases ,Membrane Proteins ,Mitochondria ,Pedigree ,Syndrome ,Mutation ,infantile hepatic mitochondrial DNA depletion ,Inner mitochondrial membrane ,Cultured ,Molecular ,DNA ,medicine.disease ,Molecular biology ,Mitochondrial ,Pair 2 ,Mitochondrial DNA depletion syndrome ,mitochondrial (mt) DNA depletion syndromes ,Human ,Cloning - Abstract
The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral1 (OMIM 251880). Known mutant genes, including TK2 (ref. 2), SUCLA2 (ref. 3), DGUOK (ref. 4) and POLG5,6, account for only a fraction of MDDS cases7. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17 (ref. 8). We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product9, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17 –/– mice.
- Published
- 2006
22. LYRM7/MZM1L is a UQCRFS1 chaperone involved in the last steps of mitochondrial Complex III assembly in human cells
- Author
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Dennis R. Winge, Jennifer L. Fox, Teresa Lobo, Ester Sánchez, Erika Fernandez-Vizarra, and Massimo Zeviani
- Subjects
Iron-Sulfur Proteins ,Mitochondrial respiratory chain ,Saccharomyces cerevisiae Proteins ,Protein subunit ,Complex III ,Saccharomyces cerevisiae ,Molecular Sequence Data ,Biophysics ,Biology ,Biochemistry ,Article ,Mitochondrial Proteins ,03 medical and health sciences ,Electron Transport Complex III ,Mice ,0302 clinical medicine ,Animals ,Humans ,Amino Acid Sequence ,Inner mitochondrial membrane ,030304 developmental biology ,Assembly factor ,chemistry.chemical_classification ,0303 health sciences ,ATPases Associated with Diverse Cellular Activities ,Apoptosis Regulatory Proteins ,HEK293 Cells ,HeLa Cells ,Molecular Chaperones ,Cell Biology ,biology.organism_classification ,Enzyme ,chemistry ,Mitochondrial matrix ,Chaperone (protein) ,Coenzyme Q – cytochrome c reductase ,biology.protein ,lipids (amino acids, peptides, and proteins) ,030217 neurology & neurosurgery - Abstract
The mammalian Complex III (CIII) assembly process is yet to be completely understood. There is still a lack in understanding of how the structural subunits are put together and which additional factors are involved. Here we describe the identification and characterization of LYRM7, a human protein displaying high sequence homology to the Saccharomyces cerevisiae protein Mzm1, which was recently shown as an assembly factor for Rieske Fe–S protein incorporation into the yeast cytochrome bc1 complex. We conclude that human LYRM7, which we propose to be renamed MZM1L (MZM1-like), works as a human Rieske Fe–S protein (UQCRFS1) chaperone, binding to this subunit within the mitochondrial matrix and stabilizing it prior to its translocation and insertion into the late CIII dimeric intermediate within the mitochondrial inner membrane. Thus, LYRM7/MZM1L is a novel human CIII assembly factor involved in the UQCRFS1 insertion step, which enables formation of the mature and functional CIII enzyme.
- Published
- 2013
23. Supercomplex assembly determines electron flux in the mitochondrial electron transport chain
- Author
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Acisclo Pérez-Martos, Raquel Moreno-Loshuertos, Pedro M. Quirós, Raquel Cruz, M. A. C. Rodríguez-Hernández, Enrique Calvo, José Antonio Enríquez, Angel Carracedo, Plácido Navas, Carmen Colás, Erika Fernandez-Vizarra, Esther Lapuente-Brun, Ana Latorre-Pellicer, Carlos López-Otín, Eduardo Balsa, Ester Perales-Clemente, Patricio Fernández-Silva, and Rebeca Acín-Pérez
- Subjects
Stereochemistry ,Ubiquinone ,Cells ,Molecular Sequence Data ,Biology ,Inbred C57BL ,Electron Transport ,Electron Transport Complex IV ,Electron Transport Complex III ,Mice ,Amino Acid Sequence ,Animals ,Cells, Cultured ,Cytochromes c ,Electron Transport Complex I ,Gene Knockdown Techniques ,HEK293 Cells ,Humans ,Mice, Inbred C57BL ,Mitochondria ,Multidisciplinary ,Cultured ,Cytochrome c ,Genetic modulation ,Electron transport chain ,Crystallography ,Electron flux ,Coenzyme Q – cytochrome c reductase ,Respirasome ,biology.protein - Abstract
Report.-- et al., The textbook description of mitochondrial respiratory complexes (RCs) views them as free-moving entities linked by the mobile carriers coenzyme Q (CoQ) and cytochrome c (cyt c). This model (known as the fluid model) is challenged by the proposal that all RCs except complex II can associate in supercomplexes (SCs). The proposed SCs are the respirasome (complexes I, III, and IV), complexes I and III, and complexes III and IV. The role of SCs is unclear, and their existence is debated. By genetic modulation of interactions between complexes I and III and III and IV, we show that these associations define dedicated CoQ and cyt c pools and that SC assembly is dynamic and organizes electron flux to optimize the use of available substrates.
- Published
- 2013
24. Tissue-specific differences in mitochondrial activity and biogenesis
- Author
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Patricio Fernández-Silva, Julio Montoya, José Antonio Enríquez, Acisclo Pérez-Martos, and Erika Fernandez-Vizarra
- Subjects
Male ,Mitochondrial DNA ,Cell type ,Transcription, Genetic ,Wistar ,Gene Dosage ,OXPHOS capacity ,mtDNA ,Tissue-specificity ,Copy number ,Animals ,Cell Nucleus ,Citrate (si)-Synthase ,DNA, Mitochondrial ,Electron Transport Complex IV ,Female ,Mitochondria ,Muscle, Skeletal ,Oxidative Phosphorylation ,Rats ,Rats, Wistar ,Gene Expression Regulation ,Organ Specificity ,Oxidative phosphorylation ,Biology ,Genome ,chemistry.chemical_compound ,Genetic ,Transcription (biology) ,Molecular Biology ,Regulation of gene expression ,Genetics ,DNA ,Skeletal ,Cell Biology ,Mitochondrial ,Cell biology ,chemistry ,Muscle ,Molecular Medicine ,Transcription ,Biogenesis - Abstract
Each cell type develops and maintains a specific oxidative phosphorylation (OXPHOS) capacity to satisfy its metabolic and energetic demands. This implies that there are differences between tissues in mitochondrial number, function, protein composition and morphology. The OXPHOS system biogenesis requires the coordinated expression of both mitochondrial and nuclear genomes. Mitochondrial DNA (mtDNA) expression can be regulated at different levels (replication, transcription, translation and post-translational levels) to contribute to the final observed OXPHOS activities. By analyzing five mammalian tissues, we evaluated the differences in the cellular amount of mtDNA and its correlation with the final observed mitochondrial activity.
- Published
- 2010
25. Isolation of mitochondria for biogenetical studies: An update
- Author
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Erika Fernandez-Vizarra, José Antonio Enríquez, Acisclo Pérez-Martos, Massimo Zeviani, Patricio Fernández-Silva, and Gustavo Ferrín
- Subjects
Mitochondrial DNA ,Isolation of mitochondria ,In organello assays ,mtDNA expression studies ,Protein import ,Animals ,Cell Fractionation ,Mice ,Mitochondria ,Rats ,Aminoacylation ,Mitochondrion ,Biology ,chemistry.chemical_compound ,Organelle ,Protein biosynthesis ,Molecular Biology ,RNA ,MtDNA expression studies ,Cell Biology ,Cell biology ,Biochemistry ,chemistry ,Molecular Medicine ,Biogenesis ,DNA - Abstract
The use of good quality preparations of isolated mitochondria is necessary when studying the mitochondrial biogenetical activities. This article explains a fast and simple method for the purification of mammalian mitochondria from different tissues and cultured cells, that is suitable for the analysis of many aspects of the organelle’s biogenesis. The mitochondria isolated following the protocol described here, are highly active and capable of DNA, RNA and protein synthesis. Mitochondrial tRNA aminoacylation, mtDNA–protein interactions and specific import of added proteins into the organelles, can also be studied using this kind of preparations.
- Published
- 2010
26. Five entry points of the mitochondrially encoded subunits in mammalian complex I assembly
- Author
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Nieves Movilla, Rebeca Acín-Pérez, Raquel Moreno-Loshuertos, María Pilar Bayona-Bafaluy, José Antonio Enríquez, Acisclo Pérez-Martos, Patricio Fernández-Silva, Erika Fernandez-Vizarra, and Ester Perales-Clemente
- Subjects
Electrophoresis ,Mitochondrial DNA ,Protein subunit ,DNA Mutational Analysis ,Molecular Sequence Data ,Mitochondrion ,Biology ,medicine.disease_cause ,MT-RNR1 ,DNA, Mitochondrial ,Models, Biological ,Cell Line ,Mice ,Models ,medicine ,Animals ,Molecular Biology ,Base Sequence ,Electron Transport Complex I ,Electrophoresis, Polyacrylamide Gel ,Mitochondria ,Mutation ,Nuclear Proteins ,Protein Subunits ,Staining and Labeling ,Genetics ,Polyacrylamide Gel ,Cell Biology ,Articles ,DNA ,Biological ,Cell biology ,Mitochondrial ,Mitochondrial respiratory chain ,Biogenesis - Abstract
Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.
- Published
- 2010
27. Early-onset liver mtDNA depletion and late-onset proteinuric nephropathy in Mpv17 knockout mice
- Author
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Marina Mora, Erika Fernandez-Vizarra, Roberto Vettor, Carlo Viscomi, Massimo Zeviani, Marco Maggioni, Claudio Pagano, Valeria Massa, and Antonella Spinazzola
- Subjects
medicine.medical_specialty ,Cirrhosis ,Transcription, Genetic ,Cells ,Knockout ,Respiratory chain ,Biology ,DNA, Mitochondrial ,Mitochondrial depletion ,Nephropathy ,Focal Segmental ,Mitochondrial Proteins ,Mice ,Focal segmental glomerulosclerosis ,Glomerulosclerosis ,Genetic ,Internal medicine ,Genetics ,medicine ,Animals ,Humans ,Age of Onset ,MPV17 ,Molecular Biology ,Cells, Cultured ,Genetics (clinical) ,Mice, Knockout ,Cultured ,Glomerulosclerosis, Focal Segmental ,Animal ,Disease Models, Animal ,Fibroblasts ,Liver ,Membrane Proteins ,Organ Specificity ,Proteinuria ,Transcription Factors ,Articles ,General Medicine ,Anatomy ,DNA ,medicine.disease ,Mitochondrial ,Endocrinology ,Knockout mouse ,Disease Models ,Transcription - Abstract
In humans, MPV17 mutations are responsible for severe mitochondrial depletion syndrome, mainly affecting the liver and the nervous system. To gain insight into physiopathology of MPV17-related disease, we investigated an available Mpv17 knockout animal model. We found severe mtDNA depletion in liver and, albeit to a lesser extent, in skeletal muscle, whereas hardly any depletion was detected in brain and kidney, up to 1 year after birth. Mouse embryonic fibroblasts did show mtDNA depletion, but only after several culturing passages, or in a serumless culturing medium. In spite of severe mtDNA depletion, only moderate decrease in respiratory chain enzymatic activities, and mild cytoarchitectural alterations, were observed in the Mpv17(-/-) livers, but neither cirrhosis nor failure ever occurred in this organ at any age. The mtDNA transcription rate was markedly increased in liver, which could contribute to compensate the severe mtDNA depletion. This phenomenon was associated with specific downregulation of Mterf1, a negative modulator of mtDNA transcription. The most relevant clinical features involved skin, inner ear and kidney. The coat of the Mpv17(-/-) mice turned gray early in adulthood, and 18-month or older mice developed focal segmental glomerulosclerosis (FSGS) with massive proteinuria. Concomitant degeneration of cochlear sensory epithelia was reported as well. These symptoms were associated with significantly shorter lifespan. Coincidental with the onset of FSGS, there was hardly any mtDNA left in the glomerular tufts. These results demonstrate that Mpv17 controls mtDNA copy number by a highly tissue- and possibly cytotype-specific mechanism.
- Published
- 2009
28. FASTKD2 nonsense mutation in an infantile mitochondrial encephalomyopathy associated with cytochrome c oxidase deficiency
- Author
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Orly Elpeleg, Pio D'Adamo, Paolo Gasparini, Daniele Ghezzi, Massimo Zeviani, Valeria Tiranti, Erika Fernandez-Vizarra, Ann Saada, Ghezzi, D, Saada, A, D'Adamo, ADAMO PIO, FERNANDEZ VIZARRA, E, Gasparini, Paolo, Tiranti, V, Elpeleg, O, and Zeviani, M.
- Subjects
Mitochondrial encephalomyopathy ,Male ,Candidate gene ,Mutant ,Nonsense mutation ,Cytochrome-c Oxidase Deficiency ,Mitochondrion ,Protein Serine-Threonine Kinases ,Mitochondrial myopathy ,Mitochondrial Encephalomyopathies ,Report ,Chlorocebus aethiops ,medicine ,Genetics ,Cytochrome c oxidase ,Animals ,Humans ,Genetics(clinical) ,Genetic Predisposition to Disease ,COS Cells ,Female ,HeLa Cells ,Infant ,Infant, Newborn ,Mitochondria ,Muscle, Skeletal ,Pedigree ,Protein-Serine-Threonine Kinases ,Siblings ,Codon, Nonsense ,Codon ,Genetics (clinical) ,biology ,Skeletal ,medicine.disease ,Newborn ,Nonsense ,biology.protein ,Muscle - Abstract
In two siblings we found a mitochondrial encephalomyopathy, characterized by developmental delay, hemiplegia, convulsions, asymmetrical brain atrophy, and low cytochrome c oxidase (COX) activity in skeletal muscle. The disease locus was identified on chromosome 2 by homozygosity mapping; candidate genes were prioritized for their known or predicted mitochondrial localization and then sequenced in probands and controls. A homozygous nonsense mutation in the KIAA0971 gene segregated with the disease in the proband family. The corresponding protein is known as fas activated serine-threonine kinase domain 2, FASTKD2. Confocal immunofluorescence colocalized a tagged recombinant FASTKD2 protein with mitochondrial markers, and membrane-potential-dependent in vitro mitochondrial import was demonstrated in isolated mitochondria. In staurosporine-induced-apoptosis experiments, decreased nuclear fragmentation was detected in treated mutant versus control fibroblasts. In conclusion, we found a loss-of-function mutation in a gene segregating with a peculiar mitochondrial encephalomyopathy associated with COX deficiency in skeletal muscle. The corresponding protein is localized in the mitochondrial inner compartment. Preliminary data indicate that FASTKD2 plays a role in mitochondrial apoptosis.
- Published
- 2008
29. In Vivo and In Organello Analyses of Mitochondrial Translation
- Author
-
Erika Fernandez-Vizarra, Patricio Fernández-Silva, Rebeca Acín-Pérez, Acisclo Pérez-Martos, and José Antonio Enríquez
- Subjects
Genetics ,Mitochondrial DNA ,Nuclear gene ,Staining and Labeling ,Mitochondrial translation ,Animals ,Humans ,In Vitro Techniques ,Mitochondria ,Mitochondrial Proteins ,Multiprotein Complexes ,Oxidative Phosphorylation ,Genetic Techniques ,Protein Biosynthesis ,Translation (biology) ,Mitochondrion ,Biology ,Cell biology ,Trypsinization ,Cell culture ,Protein biosynthesis - Abstract
Publisher Summary This chapter describes in vivo and in organello methods currently used in the laboratory to analyze the translation capacity of mitochondria from mammalian cells, both for basic research and as a tool to determine the molecular effects of mitochondrial DNA (mtDNA) mutations. Different types of cell lines can be used for the in vivo analysis of mitochondrial translation. The protocol described in the chapter is for the more commonly used anchorage-dependent transformed cells and should be adapted for each particular case (e.g., cells growing in suspension do not need to be trypsinized but are concentrated by low speed centrifugation, to collect them and/or remove the growing medium and wash the cells prior to labeling). In organello analysis of protein synthesis, the isolated mitochondria must be fully functional and able to perform coupled respiration. This requires a quick and gentle purification procedure. Mitochondria possess their own translation system devoted to the synthesis of the mtDNA-encoded polypeptides. Analysis of the protein synthesis capacity of mitochondria can be a useful tool to study basic aspects of mtDNA expression, as well as to detect defects due to mutations in the mtDNA or in nuclear genes involved in this process. The combination of the different options for labeling and analysis of the translation products must be adapted for each particular application.
- Published
- 2007
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