DOCSLIB.ORG

Metabolic Consequences of NDUFS4 Gene Deletion in Immortalized Mouse Embryonic fibroblasts☆

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Metabolic Consequences of NDUFS4 Gene Deletion in Immortalized Mouse Embryonic fibroblasts☆ View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1817 (2012) 1925–1936 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbabio Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts☆ Federica Valsecchi a,b, Claire Monge c, Marleen Forkink a, Ad J.C. de Groof c, Giovanni Benard d, Rodrigue Rossignol d, Herman G. Swarts a, Sjenet E. van Emst-de Vries a, Richard J. Rodenburg b, Maria A. Calvaruso b, Leo G.J. Nijtmans b, Bavo Heeman a, Peggy Roestenberg a, Be Wieringa c, Jan A.M. Smeitink b, Werner J.H. Koopman a,⁎, Peter H.G.M. Willems a a Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands b Department of Pediatrics, Nijmegen Centre of Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands c Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands d Institut National de la Santé et de la Recherche Médicale (INSERM), Universite Victor Segalen-Bordeaux 2, EA4576 Maladies Rares: Génétique et Métabolisme, Bordeaux, France article info abstract Article history: Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To Received 31 January 2012 better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects Received in revised form 1 March 2012 cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived Accepted 4 March 2012 from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the Available online 11 March 2012 NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activ- Keywords: ities and normal citrate synthase and CV (FoF1-ATPase) activity. Native electrophoresis of KO cell mitochon- Metabolic disorder drial fractions revealed two distinct CI subcomplexes of ~830 kDa (enzymatically inactive) and ~200 kDa Oxidative phosphorylation (active). The level of fully-assembled CII–CV was not affected by NDUFS4 gene deletion. KO cells exhibited Respirometry a moderately reduced maximal and routine O2 consumption, which was fully inhibited by acute application Live-cell microscopy of the CI inhibitor rotenone. The aberrant CI assembly and reduced O2 consumption in KO cells were fully Glycolysis normalized by NDUFS4 gene complementation. Cellular [NAD+]/[NADH] ratio, lactate production and mito- chondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP+]/[NADPH] ratio, cellular glucose consump- tion, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen spe- cies (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012). © 2012 Elsevier B.V. All rights reserved. 1. Introduction transduction and survival. Structurally, mitochondria consist of an outer membrane (MOM) that envelops a highly folded inner mem- Mitochondria generate energy in the form of adenosine triphos- brane (MIM). The latter surrounds the mitochondrial matrix com- phate (ATP) and play a crucial role in cell metabolism, signal partment where many metabolic reactions take place [1]. Proper mitochondrial functioning requires the presence of a sufficiently large proton gradient across the MIM. This gradient is maintained Abbreviations: ROS, reactive oxygen species; CI, complex I or NADH:ubiquinone ox- by the action of the electron transport chain (ETC), which consists Δψ idoreductase; , mitochondrial membrane potential; ETC, electron transport chain; of 4 multi-subunit complexes [2]. The first complex (CI) extracts elec- MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane; OXPHOS, oxidative phosphorylation; PMF, proton-motive force; ROS, reactive oxygen trons from reduced nicotinamide adenin-dinucleotide (NADH) and species; TMRM, tetramethyl rhodamine methyl ester donates them to coenzyme Q (CoQ or ubiquinone), allowing their ☆ This article is part of a special issue entitled: 17th European Bioenergetics Confer- transport to complex III (CIII). Also complex II (CII) donates electrons ence (EBEC 2012). to CoQ by liberating them from succinate. Next, electrons are trans- ⁎ Corresponding author at: 286 Biochemistry, Nijmegen Centre for Molecular Life ported by cytochrome-c (cyt-c) to complex IV (CIV) where they are Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands. Tel.: +31 24 3614589; fax: +31 24 3616413. donated to molecular oxygen (O2) to form water (H2O). At CI, CIII E-mail address: [email protected] (W.J.H. Koopman). and CIV the energy released by the electron transport is used to 0005-2728/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2012.03.006 1926 F. Valsecchi et al. / Biochimica et Biophysica Acta 1817 (2012) 1925–1936 expel protons from the mitochondrial matrix across the MIM. The lat- culture conditions to study the (patho)physiology of mitochondrial ter generates an inwardly directed trans-MIM electrochemical proton (dys)function. motive force (PMF), associated with an inside-negative mitochondrial membrane potential (Δψ), and a chemical proton gradient (ΔpH). 2. Materials and methods The PMF-driven backflow of protons is used by the FoF1-ATPase (CV) to drive mitochondrial ATP synthesis. Together, the ETC and 2.1. Generation and culturing of immortalized mouse embryonic CV constitute the oxidative phosphorylation (OXPHOS) system [3]. fibroblasts Mitochondrial CI (or NADH:ubiquinone oxidoreductase; EC 1.6.5.3) consists of 45 different subunits and constitutes the largest Primary mouse embryonic fibroblast (MEF) cultures were gener- OXPHOS complex with a molecular weight of ~1000 kDa [2,4–7]. ated from individual E12.5–13.5 embryos isolated from the inter- Seven of the CI subunits are encoded by the mitochondrial DNA cross of heterozygous NDUFS4+/− mice [25]. In brief, embryos were (mtDNA) and the remainder by nuclear DNA (nDNA). The CI catalytic dissected and the head and the internal organs were removed. The core consists of 14 evolutionary conserved “structural” subunits that, remaining tissue was minced and digested for 15 min with Trypsin- in humans, are encoded by 7 nDNA (NDUFV1, NDUFV2, NDUFS1, EDTA. This procedure was repeated until the tissue was completely NDUFS2, NDUFS3, NDUFS7, NDUFS8) and the 7 mtDNA genes (ND1– in suspension. The suspension was seeded in a T75 flask with ND6 and ND4L; [2,8]). The remainder of CI consists of 31 nDNA- DMEM medium (no. 10938, Invitrogen, Breda, The Netherlands), con- encoded supernumerary (“accessory”) subunits, of which it is pre- taining 25 mM D-glucose and supplemented with L-glutamine (4 mM sumed that they do not directly participate in the catalytic action of final concentration), 1 mM sodium pyruvate, 10% (v/v) Fetal Calf CI [2,9]. In vitro and live-cell experiments suggest that biogenesis of Serum (FCS, Greiner Bio-one, Frickenhausen, Germany) and 0.1% (v/ CI involves the stepwise assembly of pre-assembled modules v) Gentamycin (Invitrogen). The obtained cells were cultured in a hu- [5,10–13]. This process is assisted by at least 4 assembly-factors midified atmosphere of 95% air and 5% CO2 at 37 °C, expanded for two (NDUFAF1–NDUFAF4), CIII and CIV [2,6,7,14]. In humans, mutations passages and genotyped using polymerase chain reaction (PCR) anal- in mtDNA- and nDNA-encoded CI structural, assembly-factor and ac- ysis and specific forward and reverse primers designed for the cessory genes are associated with isolated CI deficiency (OMIM NDUFS4 gene: 5′-AGCCTGTTCTCATACCTCGG-3′ (forward; ~1229 bp) 252010). Currently, such mutations have been described for all 7 mi- and 5′-TTGTGCTTACAGGTTCAAAGTGA-3′ (reverse;~429 bp). DNA tochondrial mtDNA-encoded genes, all 4 known assembly factors and was isolated from MEFs using the easy-DNATM kit (Invitrogen), 17 of the nDNA-encoded genes [2,7,15]. Mutations in nDNA-encoded according to the manufacturer's instructions. The PCR was performed CI subunits are generally inherited in an autosomal recessive manner using a Phire hot start II DNA Polymerase kit (Finnzymes, Espoo, Fin- and induce CI deficiency, which is associated with a range of progres- land). The samples were heated at 98 °C (40 s). Subsequent annealing sive neurological disorders, often characterized by an early onset and and extension were carried out at 52 °C (10 s) and at 72 °C (1 min), short devastating course [16]. respectively. Next, 34 cycles were carried out at 98 °C (10 s). Final ex- The NDUFS4 gene is one of the genes often mutated in isolated tension was performed at 72 °C (10 min) on a Peltier Thermal Cycler human CI deficiency [17–24]. NDUFS4 mutations are generally associ- (MJ Research PTC-200). Using the above strategy, primary MEF cell ated with Leigh syndrome
Load more

Recommended publications
  • High-Throughput, Pooled Sequencing Identifies Mutations in NUBPL And
    ARTICLES High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency Sarah E Calvo1–3,10, Elena J Tucker4,5,10, Alison G Compton4,10, Denise M Kirby4, Gabriel Crawford3, Noel P Burtt3, Manuel Rivas1,3, Candace Guiducci3, Damien L Bruno4, Olga A Goldberger1,2, Michelle C Redman3, Esko Wiltshire6,7, Callum J Wilson8, David Altshuler1,3,9, Stacey B Gabriel3, Mark J Daly1,3, David R Thorburn4,5 & Vamsi K Mootha1–3 Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function. We established genetic diagnoses in 13 of 60 previously unsolved cases using confirmatory experiments, including cDNA complementation to show that mutations in NUBPL and FOXRED1 can cause complex I deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can be used to identify causal mutations in individual cases. Complex I of the mitochondrial respiratory chain is a large ~1-MDa ­assembly factors are probably required, as suggested by the 20 factors macromolecular machine composed of 45 protein subunits encoded necessary for assembly of the smaller complex IV9 and by cohort by both the nuclear and mitochondrial (mtDNA) genomes.
    [Show full text]
  • Proteomic and Metabolomic Analyses of Mitochondrial Complex I-Deficient
    THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 24, pp. 20652–20663, June 8, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene*□S Received for publication, November 25, 2011, and in revised form, April 5, 2012 Published, JBC Papers in Press, April 25, 2012, DOI 10.1074/jbc.M111.327601 Dillon W. Leong,a1 Jasper C. Komen,b1 Chelsee A. Hewitt,a Estelle Arnaud,c Matthew McKenzie,d Belinda Phipson,e Melanie Bahlo,e,f Adrienne Laskowski,b Sarah A. Kinkel,a,g,h Gayle M. Davey,g William R. Heath,g Anne K. Voss,a,h René P. Zahedi,i James J. Pitt,j Roman Chrast,c Albert Sickmann,i,k Michael T. Ryan,l Gordon K. Smyth,e,f,h b2 a,h,m,n3 David R. Thorburn, and Hamish S. Scott Downloaded from From the aMolecular Medicine Division, gImmunology Division, and eBioinformatics Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia, the bMurdoch Childrens Research Institute, Royal Children’s Hospital and Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the cDépartement de Génétique Médicale, Université de Lausanne, 1005 Lausanne, Switzerland, the dCentre for Reproduction and Development, Monash Institute of Medical Research, Clayton, Victoria 3168, Australia, the hDepartment of Medical Biology
    [Show full text]
  • Low Abundance of the Matrix Arm of Complex I in Mitochondria Predicts Longevity in Mice
    ARTICLE Received 24 Jan 2014 | Accepted 9 Apr 2014 | Published 12 May 2014 DOI: 10.1038/ncomms4837 OPEN Low abundance of the matrix arm of complex I in mitochondria predicts longevity in mice Satomi Miwa1, Howsun Jow2, Karen Baty3, Amy Johnson1, Rafal Czapiewski1, Gabriele Saretzki1, Achim Treumann3 & Thomas von Zglinicki1 Mitochondrial function is an important determinant of the ageing process; however, the mitochondrial properties that enable longevity are not well understood. Here we show that optimal assembly of mitochondrial complex I predicts longevity in mice. Using an unbiased high-coverage high-confidence approach, we demonstrate that electron transport chain proteins, especially the matrix arm subunits of complex I, are decreased in young long-living mice, which is associated with improved complex I assembly, higher complex I-linked state 3 oxygen consumption rates and decreased superoxide production, whereas the opposite is seen in old mice. Disruption of complex I assembly reduces oxidative metabolism with concomitant increase in mitochondrial superoxide production. This is rescued by knockdown of the mitochondrial chaperone, prohibitin. Disrupted complex I assembly causes premature senescence in primary cells. We propose that lower abundance of free catalytic complex I components supports complex I assembly, efficacy of substrate utilization and minimal ROS production, enabling enhanced longevity. 1 Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne NE4 5PL, UK. 2 Centre for Integrated Systems Biology of Ageing and Nutrition, Newcastle University, Newcastle upon Tyne NE4 5PL, UK. 3 Newcastle University Protein and Proteome Analysis, Devonshire Building, Devonshire Terrace, Newcastle upon Tyne NE1 7RU, UK. Correspondence and requests for materials should be addressed to T.v.Z.
    [Show full text]
  • Novel NDUFS4 Gene Mutation in an Atypical Late-Onset Mitochondrial Form of Multifocal Dystonia Celine Bris, Tiphaine Rouaud, Valerie Desquiret-Dumas, Et Al
    Clinical/Scientific Notes Celine Bris, PharmD NOVEL NDUFS4 GENE MUTATION IN AN enzyme activities revealed, however, a moderate Tiphaine Rouaud, MD ATYPICAL LATE-ONSET MITOCHONDRIAL FORM reduction of complex I enzyme activity in both mus- Valerie Desquiret-Dumas, OF MULTIFOCAL DYSTONIA cle and fibroblasts (figure, C). The patient was given PhD coenzyme Q10 (150 mg/d) since the age of 22 years Naig Gueguen, PhD Mitochondrial complex I, the largest component of and received routine injections of botulinum toxin David Goudenege, PhD the mitochondrial respiratory chain, comprises 44 into dystonic muscles. Magalie Barth, MD subunits of which 7 are encoded by the mitochondrial The complex I enzyme deficiency identified in Dominique Bonneau, genome and the remainder by the nuclear genome.1 muscle prompted us to look for molecular abnormal- MD, PhD Isolated complex I deficiencies represent a major con- ities. Blood sample was obtained from the patient Patrizia Amati-Bonneau, tribution within the group of respiratory chain de- after written informed consent was obtained. The MD fects.2 We report an atypical case carrying entire mitochondrial genome sequence did not reveal Guy Lenaers, PhD a homozygous NDUFS4 missense mutation, with pathogenic mutations. Thereafter, targeted rese- Pascal Reynier, MD, PhD late-onset multifocal dystonia, in contrast to expected quencing of 179 nuclear-encoded mitochondrial Anne-Sophie Lebre, clinical phenotypes due to other NDUFS4 mutations, genes, including all known structural and assembly PharmD, PhD which have been constantly reported to be responsi- complex I genes, was performed (table e-1, http:// Vincent Procaccio, MD, ble for Leigh syndrome of early onset and death.3 links.lww.com/NXG/A10), revealing a novel homo- PhD zygous c.369C.A mutation in exon 4 of NDUFS4, confirmed by Sanger sequencing, changing an evolu- Neurol Genet Case report.
    [Show full text]
  • Leigh Syndrome
    Leigh syndrome What is Leigh syndrome? Leigh syndrome is an inherited progressive neurodegenerative disease characterized by developmental delays or regression, low muscle tone, neuropathy, and severe episodes of illness that can lead to early death.1,2 Individuals with Leigh syndrome have defects in one of many proteins involved in the mitochondrial respiratory chain complexes that produce energy in cells.1 These protein defects cause lesions in tissues that require large amounts of energy, including the brainstem, thalamus, basal ganglia, cerebellum, and spinal cord.1 Clinical symptoms depend on which areas of the central nervous system are involved.1,2 Leigh syndrome is also known as subacute necrotizing encephalomyelopathy.3 What are the symptoms of Leigh syndrome and what treatment is available? Leigh syndrome is a disease that varies in age at onset and rate of progression. Signs of Leigh syndrome can be seen before birth, but are usually noticed soon after birth. Signs and symptoms may include: 2,3,4 • Hypotonia (low muscle tone) • Intellectual and physical developmental delay and regression • Digestive problems and failure to thrive • Cerebellar ataxia (difficulty coordinating movements) • Peripheral neuropathy (disease affecting nerves) • Hypertrophic cardiomyopathy • Breathing problems, leading to respiratory failure • Metabolic or neurological crisis (serious episode of illness) often triggered by an infection During a crisis, symptoms can progress to respiratory problems, seizures, coma, and possibly death. There is no cure for Leigh syndrome. Treatment is supportive. Approximately 50% of affected individuals die by three years of age, although some patients may survive beyond childhood.4 How is Leigh Syndrome inherited? Autosomal recessive Leigh syndrome is caused by mutations in at least 36 different genes,3 including FOXRED1, NDUFAF2, NDUFS4, NDUFS7, NDUFV1, COX15, SURF1 and LRPPRC.
    [Show full text]
  • Impaired Mitochondrial Complex I Function As a Candidate Driver in The
    Emmerzaal et al. Translational Psychiatry (2020) 10:176 https://doi.org/10.1038/s41398-020-0858-y Translational Psychiatry ARTICLE Open Access Impaired mitochondrial complex I function as a candidate driver in the biological stress response and a concomitant stress-induced brain metabolic reprogramming in male mice Tim L. Emmerzaal 1,2, Graeme Preston 2,3,BramGeenen 1, Vivienne Verweij1, Maximilian Wiesmann1, Elisavet Vasileiou1, Femke Grüter1, Corné de Groot1, Jeroen Schoorl1,RenskedeVeer1, Monica Roelofs1, Martijn Arts1, Yara Hendriksen1, Eva Klimars1, Taraka R. Donti4, Brett H. Graham5,EvaMorava 2,RichardJ.Rodenburg 6 and Tamas Kozicz 1,2 Abstract Mitochondria play a critical role in bioenergetics, enabling stress adaptation, and therefore, are central in biological stress responses and stress-related complex psychopathologies. To investigate the effect of mitochondrial dysfunction on the stress response and the impact on various biological domains linked to the pathobiology of depression, a novel mouse model was created. These mice harbor a gene trap in the first intron of the Ndufs4 gene (Ndufs4GT/GT mice), encoding the NDUFS4 protein, a structural component of complex I (CI), the first enzyme of the mitochondrial electron transport chain. We performed a comprehensive behavioral screening with a broad range of behavioral, physiological, and endocrine markers, high-resolution ex vivo brain imaging, brain immunohistochemistry, and multi- 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; platform targeted mass spectrometry-based metabolomics. Ndufs4GT/GT mice presented with a 25% reduction of CI activity in the hippocampus, resulting in a relatively mild phenotype of reduced body weight, increased physical activity, decreased neurogenesis and neuroinflammation compared to WT littermates. Brain metabolite profiling revealed characteristic biosignatures discriminating Ndufs4GT/GT from WT mice.
    [Show full text]
  • A Novel Mutation in NDUFS4 Causes Leigh Syndrome in an Ashkenazi Jewish Family
    J Inherit Metab Dis (2008) 31 (Suppl 2):S461–S467 DOI 10.1007/s10545-008-1049-9 SHORT REPORT A novel mutation in NDUFS4 causes Leigh syndrome in an Ashkenazi Jewish family S. L. Anderson & W. K. Chung & J. Frezzo & J. C. Papp & J. Ekstein & S. DiMauro & B. Y. Rubin Received: 15 September 2008 /Submitted in revised form: 13 November 2008 /Accepted: 19 November 2008 / Published online: 26 December 2008 # SSIEM and Springer 2008 Summary Leigh syndrome is a neurodegenerative without consanguinity with three affected children. disorder of infancy or childhood generally due to Linkage to microsatellite markers D5S1969 and mutations in nuclear or mitochondrial genes involved D5S407 led to evaluation of the complex I gene in mitochondrial energy metabolism. We performed NDUFS4, in which we identified a novel homozygous linkage analysis in an Ashkenazi Jewish (AJ) family c.462delA mutation that disrupts the reading frame. The resulting protein lacks a cAMP-dependent protein kinase phosphorylation site required for activation of Communicating editor: John Christodoulou mitochondrial respiratory chain complex I. In a Competing interests: None declared random sample of 5000 healthy AJ individuals, the References to electronic databases: Leigh syndrome: OMIM carrier frequency of the NDUFS4 mutation c.462delA 256000. NDUFS4: OMIM 602694. NDUFS4 mRNA: GenBank was 1 in 1000, suggesting that it should be considered accession # NM_002495. : : in all AJ patients with Leigh syndrome. S. L. Anderson J. Frezzo B. Y. Rubin (*) Department of Biological Sciences, Fordham University, Abbreviations 441 E. Fordham Rd., Bronx, NY 10458, USA AJ Ashkenazi Jewish e-mail: [email protected] BCS1L BCS1-like protein W.
    [Show full text]
  • Accessory Subunits Are Integral for Assembly and Function of Human Mitochondrial Complex I
    Accessory subunits are integral for assembly and function of human mitochondrial complex I David A. Stroud1*, Elliot E. Surgenor1, Luke E. Formosa1,2, Boris Reljic2†, Ann E. Frazier3,4, Marris G. Dibley1, Laura D. Osellame1, Tegan Stait3, Traude H. Beilharz1, David R. Thorburn3-5, Agus Salim6, Michael T. Ryan1* 1Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, 3800, Melbourne, Australia. 2Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University 3086, Melbourne, Australia. 3Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne 3052, Australia 4Department of Pediatrics, University of Melbourne, Melbourne 3052, Australia. 5Victorian Clinical Genetics Services, Royal Children’s Hospital 3052, Melbourne, Australia. 6Department of Mathematics and Statistics, La Trobe University 3086, Melbourne Australia. *Correspondence to: [email protected] and [email protected] †Current address: Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain (RC) and is composed of 44 different subunits in humans, making it one of the largest known multi-subunit membrane protein complexes1. Complex I exists in supercomplex forms with RC complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP2. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson’s disease and aging3-5. Bacterial and human complex I share 14 core subunits essential for enzymatic function, however the role and requirement of the remaining 31 human accessory subunits is unclear1,6.
    [Show full text]
  • Analysis of Human Mutations in the Supernumerary Subunits of Complex I
    life Review Analysis of Human Mutations in the Supernumerary Subunits of Complex I Quynh-Chi L. Dang, Duong H. Phan, Abigail N. Johnson, Mukund Pasapuleti, Hind A. Alkhaldi, Fang Zhang and Steven B. Vik * Department of Biological Sciences, Southern Methodist University, Dallas, TX 75287, USA; [email protected] (Q.-C.L.D.); [email protected] (D.H.P.); [email protected] (A.N.J.); [email protected] (M.P.); [email protected] (H.A.A.); [email protected] (F.Z.) * Correspondence: [email protected] Received: 30 October 2020; Accepted: 16 November 2020; Published: 20 November 2020 Abstract: Complex I is the largest member of the electron transport chain in human mitochondria. It comprises 45 subunits and requires at least 15 assembly factors. The subunits can be divided into 14 “core” subunits that carry out oxidation–reduction reactions and proton translocation, as well as 31 additional supernumerary (or accessory) subunits whose functions are less well known. Diminished levels of complex I activity are seen in many mitochondrial disease states. This review seeks to tabulate mutations in the supernumerary subunits of humans that appear to cause disease. Mutations in 20 of the supernumerary subunits have been identified. The mutations were analyzed in light of the tertiary and quaternary structure of human complex I (PDB id = 5xtd). Mutations were found that might disrupt the folding of that subunit or that would weaken binding to another subunit. In some cases, it appeared that no protein was made or, at least, could not be detected. A very common outcome is the lack of assembly of complex I when supernumerary subunits are mutated or missing.
    [Show full text]
  • Mitochondrial Complex I Inhibition Is Not Required for Dopaminergic Neuron Death Induced by Rotenone, MPP؉, Or Paraquat
    Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP؉, or paraquat Won-Seok Choi*, Shane E. Kruse†, Richard D. Palmiter†‡, and Zhengui Xia*§ *Toxicology Program, Department of Environmental and Occupational Health Sciences, Graduate Program in Neurobiology and Behavior, Institute for Stem Cell and Regenerative Medicine and †Howard Hughes Medical Institute and Department of Biochemistry, University of Washington, Seattle, WA 98195 Contributed by Richard D. Palmiter, August 4, 2008 (sent for review February 12, 2007) Inhibition of mitochondrial complex I is one of the leading hypoth- (26). This causes a frame shift that precludes synthesis of the eses for dopaminergic neuron death associated with Parkinson’s mature Ndufs4. Similar deletion mutations in the human disease (PD). To test this hypothesis genetically, we used a mouse NDUFS4 gene cause deficiencies in complex I activity and infant strain lacking functional Ndufs4, a gene encoding a subunit re- lethality (27, 28). If complex I inhibition is obligatory for quired for complete assembly and function of complex I. Deletion selective dopaminergic neuron loss associated with PD, and of the Ndufs4 gene abolished complex I activity in midbrain more specifically in the rotenone, MPPϩ, and paraquat models mesencephalic neurons cultured from embryonic day (E) 14 mice, of PD, one might expect greater baseline apoptosis in cultured but did not affect the survival of dopaminergic neurons in culture. dopaminergic neurons from Ndufs4Ϫ/Ϫ mice. In addition, Although dopaminergic neurons were more sensitive than other Ndufs4Ϫ/Ϫ dopaminergic neurons should be less sensitive to .neurons in these cultures to cell death induced by rotenone, MPP؉, toxicity induced by rotenone, MPPϩ, or paraquat or paraquat treatments, the absence of complex I activity did not protect the dopaminergic neurons, as would be expected if these Results compounds act by inhibiting complex 1.
    [Show full text]
  • Mitochondrial DNA (Mtdna) Test Requisition
    SHIP TO: Medical Genetics Laboratories BCM-MEDICAL GENETICS LABORATORIES Baylor College of Medicine PHONE: 800-411-GENE | FAX: 713-798-2787 | www.bcmgeneticlabs.org 2450 Holcombe, Grand Blvd. -Receiving Dock Houston, TX 77021-2024 MITOCHONDRIAL DNA (mtDNA) TEST REQUISITION Phone: 713-798-6555 PATIENT INFORMATION INDICATION FOR STUDY NAME: DATE OF COLLECTION: / / LAST NAME FIRST NAME MI MM DD YY HOSPITAL#: ACCESSION#: DATE OF BIRTH: / / GENDER (Please select one): FEMALE MALE MM DD YY SAMPLE TYPE (Please select one): ETHNIC BACKGROUND (Select all that apply): UNKNOWN BLOOD AFRICAN AMERICAN SKELETAL MUSCLE ASIAN DNA (Specify Source): ASHKENAZIC JEWISH EUROPEAN CAUCASIAN -OR- HISPANIC OTHER (Specify): NATIVE AMERICAN INDIAN PLACE PATIENT STICKER HERE OTHER JEWISH OTHER (Please specify): REPORTING INFORMATION ADDITIONAL PROFESSIONAL REPORT RECIPIENTS PHYSICIAN: NAME: INSTITUTION: PHONE: *FAX: PHONE: *FAX: NAME: EMAIL (INTERNATIONAL CLIENT REQUIREMENT): PHONE: *FAX: *BCM-MEDICAL GENETIC LABORATORIES HAS A FAX ONLY POLICY FOR REPORTING INDICATION FOR STUDY SYMPTOMATIC (Summarize below.): *FAMILIAL MUTATION/VARIANT ANALYSIS: Complete all fields below and attach the proband's report. GENE NAME: ASYMPTOMATIC/POSITIVE FAMILY HISTORY: (ATTACH FAMILY HISTORY) MUTATION/UNCLASSIFIED VARIANT: RELATIONSHIP TO PROBAND: THIS INDIVIDUAL IS CURRENTLY: SYMPTOMATIC ASYMPTOMATIC *If family mutation is known, complete the FAMILIAL MUTATION/ VARIANT ANALYSIS section. NAME OF PROBAND: ASYMPTOMATIC/POPULATION SCREENING RELATIONSHIP TO PROBAND: OTHER (Specify clinical findings below.): BCM LAB#: A COPY OF ORIGINAL RESULTS ATTACHED IF PROBAND TESTING WAS PERFORMED AT ANOTHER LAB, CALL TO DISCUSS PRIOR TO SENDING SAMPLE. A POSITIVE CONTROL MAY BE REQUIRED IN SOME CASES. REQUIRED: NEW YORK STATE PHYSICIAN SIGNATURE OF CONSENT I certify that the patient specified above and/or their legal guardian has been informed of the benefits, risks, and limitations of the laboratory test(s) requested.
    [Show full text]
  • Leigh Syndrome Associated with Mitochondrial Complex I Deficiency Due to a Novel Mutation in the NDUFS1 Gene
    OBSERVATION Leigh Syndrome Associated With Mitochondrial Complex I Deficiency Due to a Novel Mutation in the NDUFS1 Gene Miguel A. Martín, PhD; Alberto Blázquez, BSc; Luis G. Gutierrez-Solana, MD; Daniel Fernández-Moreira, PharmB; Paz Briones, PhD; Antoni L. Andreu, MD, PhD; Rafael Garesse, PhD; Yolanda Campos, PhD; Joaquín Arenas, PhD Background: Mutations in the nuclear-encoded sub- Results: Muscle biochemistry results showed a com- units of complex I of the mitochondrial respiratory chain plex I defect of the mitochondrial respiratory chain. Se- are a recognized cause of Leigh syndrome (LS). Re- quencing analysis of the mitochondrial DNA–encoded cently, 6 mutations in the NDUFS1 gene were identified ND genes, the nuclear DNA–encoded NDUFV1, NDUFS1, in 3 families. NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, and NDUFAB1 genes, and the complex I assembly factor CIA30 Objective: To describe a Spanish family with LS, com- gene revealed a novel homozygous L231V mutation plex I deficiency in muscle, and a novel mutation in the (c.691C→G) in the NDUFS1 gene. The parents were het- NDUFS1 gene. erozygous carriers of the L231V mutation. Design: Using molecular genetic approaches, we iden- Conclusions: Identifying nuclear mutations as a cause tified the underlying molecular defect in a patient with of respiratory chain disorders will enhance the possibil- LS with a complex I defect. ity of prenatal diagnosis and help us understand how mo- lecular defects can lead to complex I deficiency. Patient: The proband was a child who displayed the clini- cal
    [Show full text]