PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/90829 Please be advised that this information was generated on 2021-09-29 and may be subject to change. Lo st in translation : genetic defects underlying combined OXPHOS complex I, III and IV deficiencies Paulien Smits Lost in translation: genetic defects underlying combined OXPHOS complex I, III and IV deficiencies Thesis Radboud University Nijmegen with a summary in Dutch © 2011 Smits, Paulien, Nijmegen, The Netherlands The research presented in this thesis was performed at the Department of Pediatrics, Radboud University Nijmegen Medical Centre, The Netherlands. ISBN 978-94-6108-189-6 Cover: Representation of the main elements and processes required for the biogenesis of the OXPHOS complexes: nuclear and mitochondrial DNA, proteins synthesized in the cytoplasm (which are components of the OXPHOS system or proteins needed for mitochondrial translation), and mitochondrial translation. At all the different steps the 'translation' can go wrong, with an A being decoded into for instance an a, in the end resulting in an OXPHOS system deficiency. To make sure we will not be lost in translation, the puzzle of combined OXPHOS complex I, III and IV deficiencies needs to be solved. Cover concept: Jordy van Emden & Paulien Smits Cover design & lay-out: Paul van Hoogstraten Printed by: Gildeprint Drukkerijen Lo st in translation : genetic defects underlying combined OXPHOS complex I, III and IV deficiencies Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het college van decanen in het openbaar te verdedigen op vrijdag 2 september 2011 om 12.30 uur precies door Paulien Smits geboren op 3 augustus 1981 te Krimpen aan den IJssel Promotoren: Prof. dr. L.P.W.J. van den Heuvel (UZ Leuven) Prof. dr. J.A.M. Smeitink Manuscriptcommissie: Prof. dr. M.A. Huynen (voorzitter) Prof. dr. G.J.M. Pruijn Prof. dr. N.V.A.M. Knoers-van Slobbe (UMC Utrecht) Geluk is het enige dat zich vermenigvuldigt als je het deelt. Albert Schweitzer Contents C o n ten ts Abbreviations 8 Chapter 1 General introduction and outline of this thesis 11 Chapter 2 Mitochondrial translation and beyond: processes implicated 19 in combined oxidative phosphorylation deficiencies Chapter 3 Functional consequences of mitochondrial tRNA Trp and 69 tRNA Arg mutations causing combined OXPHOS defects Chapter 4 Distinct clinical phenotypes associated with a mutation in 87 the mitochondrial translation elongation factor EFTs Chapter 5 Mutation in subdomain G' of mitochondrial elongation 107 factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle Chapter 6 Reconstructing the evolution of the mitochondrial 121 ribosomal proteome Chapter 7 Sequence variants in four candidate genes (NIPSNAP1, 161 GbAs , CHCHD1 and METT11D1) in patients with combined oxidative phosphorylation system deficiencies Chapter 8 Mutation in mitochondrial ribosomal protein MRPS22 leads 175 to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy Chapter 9 General discussion and future perspectives 193 Summary 219 Samenvatting 225 Samenvatting voor leken 229 Dankwoord / Acknowledgements 237 Curriculum Vitae 243 List of publications 247 List of abbreviations List of A bbreviations ADP adenosine diphosphate ATP adenosine triphosphate BN blue native bp base pair BLAST basic local alignment search tool cDNA complementary DNA CI - CV complex I - complex V CoQ coenzyme Q or ubiquinone CO(X) cytochrome c oxidase CS citrate synthase CSF cerebrospinal fluid cyt cytochrome DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate ECG electrocardiogram EEG electroencephalogram EST expressed sequence tag FCS fetal calf serum gDNA genomic DNA GDP guanosine diphosphate GTP guanosine triphosphate HSP heavy strand promoter / hereditary spastic paraplegia / heat shock protein IGA in-gel activity kDa kilo Dalton LSP light strand promoter LSU large subunit MELAS mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes MERRF myoclonic epilepsy with ragged-red fibers MLASA mitochondrial myopathy and sideroblastic anemia MLPA multiplex ligation-dependent probe amplification MRI magnetic resonance imaging mRNA messenger RNA MRP mitochondrial ribosomal protein mt mitochondrial mtDNA mitochondrial DNA 8 List of abbreviations NAD(H) nicotinamide adenine dinucleotide (reduced) ND NADH dehydrogenase or NADH CoQ oxidoreductase nDNA nuclear DNA NTP nucleoside triphosphate ORF open reading frame OXPHOS oxidative phosphorylation PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PSI-BLAST position specific iterative BLAST RNA ribonucleic acid RP ribosomal protein rRNA ribosomal RNA RT-PCR reverse transcriptase PCR SD standard deviation SDS sodium dodecyl sulphate SNP single nucleotide polymorphism SSU small subunit TIM translocase of the inner mitochondrial membrane TOM translocase of the outer mitochondrial membrane tRNA transfer RNA VEP visual evoked potential 9 If you want to see a rainbow, you have got to put up with the rain. Dolly Parton C h apter 1 General introduction and outline of this thesis H B Chapter 1 Energy is fundamental to life. For all our actions, from running to sleeping, and for the myriad of processes occurring in and outside our cells every second, energy is required. This energy is derived from food, mainly from carbohydrates and fat, and is generated in the form of adenosine triphosphate (ATP) during the following processes: glycolysis (i.e., the breakdown of glucose into pyruvate), the Krebs or citric acid cycle (i.e., breakdown of acetyl coenzyme A, which is synthesized from pyruvate, fatty acids or in lesser amounts from amino acids), and oxidative phosphorylation (Figure 1). Glycolysis takes place in the cell's cytoplasm, also in the absence of oxygen, whereas both the Krebs cycle and oxidative phosphorylation are aerobic processes occurring in the mitochondrion. Oxidative phosphorylation is by far the largest contributor to the ATP supply. M itochondria Mitochondria, often called the powerhouses of the cell, are semiautonomous organelles present in varying numbers per cell, depending on the tissue type: from zero mitochondria in red blood cells to over a thousand in cells with high energy demands. Mitochondrial morphology ranges from small bean-shaped structures to highly branched tubular networks due to constant remodeling by fusion and fission. Reminiscent of its endosymbiotic origin, i.e., that it evolved from a bacterium that was engulfed by a host cell over a billion years ago, the mitochondrion is surrounded by a double membrane and has its own, circular genome. The outer mitochondrial membrane separates the cytosol of the cell from the intermembrane space, and the highly invaginated inner membrane envelops the mitochondrial matrix, which contains the mitochondrial genome and enzymes of the Krebs cycle, among others. Most genes from the prokaryotic genome have been lost or have been transferred to the nucleus of the eukaryotic cell during evolution. In human cells, nearly all genes that are required for mitochondrial functioning reside in the nuclear genome (nDNA); mitochondrial DNA (mtDNA) merely contains 37 genes, coding for rRNAs, tRNAs and several constituents of the oxidative phosphorylation (OXPHOS) system. OXPHOS SYSTEM The respiratory or electron transport chain is composed of four enzymatic complexes and two electron carriers, ubiquinone and cytochrome c, and together with a fifth complex it makes up the OXPHOS system (Figure 1).1 The five complexes of the OXPHOS system are multiprotein structures, consisting of 89 proteins in total, and constitute the only metabolic pathway that is under dual genetic control. The OXPHOS system is embedded in the inner mitochondrial membrane and executes the final biochemical steps in energy production. NADH 12 General introduction and outline of this thesis and succinate, produced during glycolysis, fatty acid oxidation and the Krebs cycle, are oxidized by complex I and complex II, respectively, of the OXPHOS system. The hereby liberated electrons are transferred via the electron carriers and complex III to complex IV, where they are delivered to the final electron acceptor oxygen, reducing it to water. The energy released during electron transport is utilized by complexes I, III and IV to translocate protons across the mitochondrial inner membrane, generating an electrochemical proton gradient. This gradient serves as the driving force for the backflow of protons from the intermembrane space to the matrix compartment via complex V, ATP synthase. Complex V harnesses the free energy to phosphorylate ADP, using inorganic phosphate (Pi), to form ATP. CYTOSOL I Compiei; 1 1 Il iv] v| Carbohydrates Lactate I Compte» I [Compta | Compia* Figure 1. The mitochondrial oxidative phosphorylation system. Schematic representation of oxidative phosphorylation and other metabolic processes, adapted from ref. 2. Thick arrows represent the direction of electron movement and dotted arrows indicate the proton flux. For each OXPHOS complex, the number of subunits encoded by the nDNA or mtDNA is depicted at the top. The import complexes needed