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Identification and characterisation of new factors and mechanisms regulating human cytochrome c oxidase biogenesis 10.17863/CAM.39069 Signes Marrahi, Alba Zeviani, Massimo 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. University of Cambridge 2019-05-18 Thesis Doctoral Doctor of Philosophy (PhD) en https://www.repository.cam.ac.uk/handle/1810/291912 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a70b2714-b1ae-412e-8349-b859e165e816/download c5890b2f704d82e0723e588cec50c344 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b5e53f7f-704d-41c6-8b5b-6dc3c1f8c386/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Cytochrome c oxidase assembly APOPT1 MR-1S COX complex IV assembly COX assembly factors