Hughes Hall College Identification and characterisation of new factors and mechanisms regulating human cytochrome c oxidase biogenesis Alba Signes Marrahi Dissertation submitted for the degree of Doctor of Philosophy November 2018 Preface Declaration This dissertation describes the results of my own work, except for the experiments performed by collaborators, which are specified in the figure legends. All the work was carried out under the supervision of Prof. Massimo Zeviani assisted by Dr. Erika Fernandez-Vizarra (Senior Investigator Scientist) at the Medical Research Council (MRC) Mitochondrial Biology Unit, between January 2015 and November 2018. This thesis has not been submitted, in whole or in part, for a degree at this or any other institution and the length of it does not exceed the prescribed word limit. Part of the text in Chapter 1 has been published in a review article: Assembly of mammalian oxidative phosphorylation complexes I–V and supercomplexes (2018) (Signes & Fernandez-Vizarra, Essays in Biochemistry, 62(3): 255–270). The results described in Chapter 3 led to a publication: MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase (2017) (Vidoni et al., Cell reports, 18 (7), 1727-1738). The results described in Chapter 4 and 5 led to a publication: APOPT1/COA8 assists COX assembly and is oppositely regulated by UPS and ROS (2019) (Signes et al., EMBO Molecular Medicine, 11(1), e9582). Alba Signes Marrahi, Cambridge (UK), November 2018. iii Preface Acknowledgements Firstly, I would like to thank my supervisor, Prof. Massimo Zeviani, for giving me the opportunity to carry out my work in his laboratory. I also want to give a huge thank you to Dr. Erika Fernandez-Vizarra for her guidance, enthusiasm and support during the past four years. Her passion for science is inspiring. A big thank you to every single member of the Mitochondrial Medicine group, including those that already left, because no matter how busy they were, they always gave me their invaluable guidance and help (as scientists and as friends). Thanks also to all the many colleagues in the unit who were always kind and supportive with me. Special thanks to Massimo and Erika for proofreading my thesis and giving me constructive feedback. I must also thank Dr. Carlo Viscomi, Raffaele Cerutti, Prof. Mike Murphy, Dr. Liz Hinchy, Prof. James Nathan and Anna Dickson, who greatly contributed to this project. I must also acknowledge the team from the Phenomics Laboratory, Forvie Site (Cambridge Biomedical Campus, Cambridge CB2 0PY) for their fantastic work and help with animal models. A special thank you to Penny, Janet, Irina, Jennifer, Dave, Steve, Merlin, Ash, and Andrew for their help with student, administrative, technical and IT matters. And a big thanks to the Medical Research Council for funding my research experience. I have been very lucky to meet wonderful people in Cambridge, many of whom have become very close friends. A very special thank you is reserved to Nicole, who gave me her kind support and advice and was always there when needed. Lastly, I want to thank my family for their love and support and for giving me the strength and motivation that I, sometimes, lack. iv Preface Summary Assembly of the mitochondrial complex IV (CIV) or cytochrome c oxidase (COX) is an intricate and highly regulated process in which the three-core mitochondrial DNA (mtDNA) encoded subunits assemble in a coordinated way with the remaining eleven supernumerary nuclear DNA (nDNA) encoded subunits. This process requires a large number of additional factors, which are necessary for the correct maturation of the complex but are not part of the fully assembled enzyme. Studies in mutant strains of the yeast Saccharomyces cerevisiae have been very useful to find many assembly factors and their human orthologs. However, it has become evident that there are animal-specific factors not present in yeast, which need to be identified using other techniques. In this work, two of these COX assembly factors, identified through two different approaches, have been characterised. First, quantitative proteomic analysis of the subassemblies accumulated in a MT-CO3 deficient cell line allowed the identification of MR-1S, conserved only in vertebrates. The downregulation of this protein produced a COX assembly and enzymatic defect. In addition, it was found to interact with the highly conserved bona fide COX assembly factors PET100 and PET117. Secondly, genomic screening of patients displaying mitochondrial encephalopathy and COX deficiency, revealed the presence of pathogenic variants in APOPT1. An Apopt1 knockout (KO) mouse model was generated by CRISPR/Cas9 to study the role of the APOPT1 protein in relation with COX biogenesis. Phenotypic characterisation showed COX deficiency in all tissues, associated with neuromuscular impairment, similar to the features found in human individuals carrying mutations in APOPT1, for which two immortalised skin fibroblast cell lines were studied. All the analysed mouse tissues and human cells showed decreased levels of fully assembled COX and subassembly accumulation. Interestingly, APOPT1 was found to be tightly regulated at the post-translational level, being its turnover controlled by the cytoplasmic ubiquitin- proteasome system (UPS), while increased oxidative stress had stabilising effects on the mature intramitochondrial form, which was shown to protect COX subunits from oxidatively-induced degradation. v Preface Abbreviations AAVs adeno-associated virus ACO2 mitochondrial aconitase ADP adenosine diphosphate AIF apoptosis inducing factor ALS amyotrophic lateral sclerosis ANT adenine nucleotide translocator AOX alternative oxidase ATP adenosine triphosphate bp base pairs BN-PAGE Blue Native Polyacrylamide Gel Electrophoresis BSA bovine serum albumin CI complex I CII complex II CIII complex III CIV complex IV CV complex V CLAMS comprehensive laboratory animal monitoring system CNS central nervous system COX cytochrome c oxidase CT computed tomography CS citrate synthase D-loop displacement loop DDM n-dodecyl-β-D-maltoside DMEM dulbecco’s modified eagle’s medium DNA deoxyribonucleic acid DUBs deubiquitinating enzymes EM electron microscopy ER endoplasmic reticulum ERAD ER–associated protein degradation ETC electron transport chain EV empty vector FAD flavin adenine dinucleotide FBS foetal bovine serum FMN flavin mononucleotide FVB friend virus B g gram GSH glutathione (reduced) GSSG glutathione (oxidised) HA hemagglutinin HEK human embryonic kidney HIF-1α hypoxia-inducible factor-1α vi Preface HS heavy strand IGA in gel activity IHC immunohistochemistry IMM inner mitochondrial membrane IMS inter-membrane space ISC iron-sulphur cluster kDa kilodalton KGDH α-ketoglutarate dehydrogenase KO knockout LACE1 ATPase lactation elevated 1 l litre LS light strand; leigh syndrome M molar mM millimolar MAD mitochondrial-associated degradation MAVS mitochondrial antiviral signalling MCIA mitochondrial complex I assembly MCU mitochondrial calcium uniporter MEFs mouse embryo fibroblasts MELAS mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes MIA mitochondrial intermembrane space assembly machinery MitoPQ mitoparaquat MITRAC mitochondrial translation regulation assembly intermediate of cytochrome c oxidase ml millilitre MOMP mitochondrial outer membrane permeabilisation MPT mitochondrial permeability transition MR-1S/M/L myofibrillary-related protein 1 short/medium/long isoform MRI magnetic resonance imaging MRS MR-spectroscopy MS mass spectrometry Mt mitochondria mtDNA mitochondrial DNA MTS mitochondrial targeting signal MW molecular weight NAD nicotinamide adenine dinucleotide NBF neutral buffered formalin nDNA nuclear DNA NGS next-generation sequencing NMD nonsense-mediated mRNA decay OAA oxaloacetic acid OH origin of the heavy strand vii Preface OMM outer mitochondrial membrane OXPHOS oxidative phosphorylation PBS phosphate buffered saline PCR polymerase chain reaction PDHC pyruvate dehydrogenase complex PET positron emission tomography Pi inorganic phosphate PLS pump loading site PMF proton-motive force PNKD paroxysmal non-kinesigenic dyskinesia Q, CoQ ubiquinone or coenzyme Q RNA ribonucleic acid rRNA ribosomal RNA RET reverse electron transfer SDH succinate dehydrogenase SDS-PAGE sodium dodecyl sulphate-polyacrylamide polyacrylamide gel electrophoresis shRNA small hairpin RNA SILAC stable isotope-labelled amino acids in cell culture SOD2/MnSOD superoxide dismutase 2 TCA tricarboxylic acid cycle TIM transporter of the inner membrane TOM translocase of the outer membrane Tm melting temperature tRNAs transfer RNAs UPR unfolded protein response UPS ubiquitin-proteasome system WB Western blot WT Wild-type μg microgram μl microlitre μM micromolar viii Table of contents Preface Declaration ......................................................................................................... iii Acknowledgements ............................................................................................ iv Summary ............................................................................................................ v Abbreviations ...................................................................................................... vi Chapter 1: Introduction 1.1 General introduction to mitochondrial biology ....................................... 2 1.1.1 Mitochondrial origin ................................................................................ 2 1.1.2 Mitochondrial architecture